ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1115

Novel Procedures for Identificationand Characterization of ViralProteases Inhibitors

ANGELICA EHRENBERG

ISSN 1651-6214ISBN 978-91-554-8855-0urn:nbn:se:uu:diva-215700

Dissertation presented at Uppsala University to be publicly examined in B42, BMC, UppsalaUniversitet, Husargatan 3, 751 23, Uppsala, Friday, 28 February 2014 at 13:15 for the degreeof Doctor of Philosophy. The examination will be conducted in English. Faculty examiner:Professor Celia Schiffer ( University of Massachusetts Medical School).

AbstractEhrenberg, A. 2014. Novel Procedures for Identification and Characterization of ViralProteases Inhibitors. Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1115. 50 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-8855-0.

Viral proteases are often considered to be attractive drug targets because of their crucial functionin the viral replication machinery. In order to increase our knowledge of these important targetsand to contribute to the discovery and development of new antiviral drugs, the proteases fromhepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and theirinteractions with inhibitors and fragments have been characterized, using enzyme inhibition andSPR biosensor based interaction assay.

The structure activity relationships and the resistance profiles of a series of HCV NS3 proteaseinhibitors based on either P2 proline or phenylglycine residues were analyzed using wild typegenotype 1a and the major resistant variants A156T and D168V. The observed susceptibility tosubstitutions associated with these resistance variants was concluded to depend on the P2 and theP1 residue, and not only on the P2 residue as previously had been suggested. In order to be able toevaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluatedon wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155Kfrom genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, thecompounds were analyzed under conditions optimized for each variant. VX-950 was found tobe the least susceptible compound to resistance and genetic variation. A more detailed analysisshowed that the kinetic and mechanistic features of the inhibitors were significantly differentfor the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 wasrevealed to have favorable kinetics for all three genotypes. This is an advantage for the designof broad spectrum drugs.

A fragment based procedure for identifying and validating novel scaffolds for inhibitors ofHCMV protease was established. It identified fragments that may serve as starting points forthe discovery of effective inhibitors against this challenging target.

The procedures developed for the evaluation and identification of novel HCV NS3 andHCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitorinteractions that is expected to have an impact on the design of novel antiviral drugs.

Keywords: drug discovery, viral proteases, inhibitors, Hepatitis C, NS3, cytomegalovirus

Angelica Ehrenberg, Department of Chemistry - BMC, Biochemistry, Box 576, UppsalaUniversity, SE-75123 Uppsala, Sweden.

© Angelica Ehrenberg 2014

ISSN 1651-6214ISBN 978-91-554-8855-0urn:nbn:se:uu:diva-215700 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-215700)

Till Pavel, David och Viktor

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Örtqvist P., Vema A., Ehrenberg E. A., Dahl G., Åkerblom E.,

Danielson U. H. and Sandström A. Structure-Activity Rela-tionships of HCV NS3 Protease Inhibitors Evaluated on the Drug-Resistant Mutants A156T and D168A. Antiviral Thera-py, 2010; 15:841-852

II Ehrenberg E. A., Schmuck B., Anwar M. I., Gustafsson S. S.

and Danielson U. H. Characterization of hepatitis C NS3 protease inhibitors: Accounting for strain variations and re-sistance mutations (In press 2013)

III Gustafsson S. S., Ehrenberg E. A., Schmuck B., Anwar M. I. and Danielson U. H. Identification of weak points of hepatitis C virus NS3 protease inhibitors using SPR biosensor-based interaction kinetic analysis (Submitted 2013)

IV Lindgren M. T., Brandt P., Stenberg G., Ehrenberg E. A., Win-quist J., Hoffmann I., Geitmann M., Källblad P., Hämäläinen M. D. and Danielson U. H. Challenges in the discovery of fragments targeting human cytomegalovirus protease (Man-uscript)

Reprints were made with permission from the respective pub-lishers.

Publications not included in this thesis

V Lampa A., Ehrenberg E. A., Vema A., Åkerblom E., Lindeberg G., Karlén A., Danielson U. H. and Sandström A. Improved P2 Phenylglycine-based Hepatitis C Virus NS3 Protease Inhibi-tors with Alkenylic Primeside Substituents (Bioorganic Med-ical Chemistry, 2010;18(14):5413-24)

VI Örtqvist P., Gising J., Ehrenberg E. A., Vema A., Borg A., Lar-

hed M., Danielson U.H. and Sandström A. Discovery of Achi-ral Inhibitors of the Hepatitis C Virus NS3 Protease based on 2(1H)-Pyrazinones Bioorganic Medical Chemistry. 2010 18(17):6512-25

VII Gising J., Belfrage A. K., Alogheli H., Ehrenberg E. A., Åker-blom E., Mastej M., Svensson R., Artursson P., Karlén A., Da-nielson U. H., Larhed M. and Sandström A. Achiral Pyrazi-none-Based Inhibitors of the Hepatitis C Virus NS3 Prote-ase and Drug-Resistant Variants with Elongated Substitu-ents Directed Toward the S2 Pocket. (Journal of Medical Chemistry. 2013)

VIII Lampa A, Ehrenberg E. A., Vema A., Åkerblom E., Lindeberg G., Danielson U. H., Karlén A. and Sandström A. P2-P1’ Mac-rocyclization of P2 Phenylglycine based HCV NS3 Protease Inhibitors using Ring-closing Metathesis. Bioorg Med Chem. 2011; 19(16):4917-27

IX Lampa, A., Grandin J., Ehrenberg E. A., Alogheli, H.,

Åkerblom, E., Lindeberg, G., Danielson, U. H., Karlén, A., Sandstöm, A. Diversely Vinylated Acyclic Pyrimidinyloxy-phenylglycine-based Inhibitors of the HCV NS3 Protease and Corresponding Macrocycles- Beneficial use of an Aro-matic P1 Moiety. (Manuscript)

Contents

Introduction ................................................................................................... 11 Antiviral drug discovery ........................................................................... 12

Target validation .................................................................................. 12 Assay development .............................................................................. 13 Hit identification .................................................................................. 13 Lead optimization ................................................................................ 13

Viral evolution .......................................................................................... 14 Genetic variation of HCV .................................................................... 14 Drug resistance .................................................................................... 15

Viral proteases as drug targets .................................................................. 16 Hepatitis C virus NS3 protease ............................................................ 17 Human cytomegalovirus protease ........................................................ 22

Evaluation of protease inhibitors .............................................................. 23 Brief introduction to Michealis-Menten kinetics ................................. 23 Interaction analysis .............................................................................. 26 Inhibition and interaction data and their relevance for drug design .... 28

Present investigation ..................................................................................... 29 Aim ........................................................................................................... 29 Production of proteases ............................................................................ 29

HCV protease variants ......................................................................... 29 HCMV protease variants ..................................................................... 30

Interaction studies .................................................................................... 30 Structure-activity analysis of HCV NS3 inhibitors (Paper I) ................... 30

Conclusion ........................................................................................... 32 Evaluation of HCV NS3 inhibitors (paper II) .......................................... 33

Conclusions ......................................................................................... 35 Detailed characterization of NS3 inhibitors (Paper III) ........................... 35

Conclusions ......................................................................................... 38 Fragment-based discovery of HCMV protease inhibitors (paper IV) ...... 39

Conclusions ......................................................................................... 41

Conclusions and future perspectives ............................................................. 42 Conclusions .............................................................................................. 42 Future perspectives ................................................................................... 42

Svensk sammanfattning ................................................................................ 43 Utveckling av läkemedel mot virala sjukdomar ....................................... 43

Min forskning ........................................................................................... 43

Acknowledgments......................................................................................... 45

References ..................................................................................................... 48

Abstract

Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay. The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on enzyme variants that differ catalytically, the compounds were analyzed under condi-tions optimized for each enzyme variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs. A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target. The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have been found to be efficient and informative. Overall, the work has contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs.

Abbreviations

HCV HCMV HIV AIDS FDA DAA FBDD HTS SAR FRET NMR SPR NS3 DABCYL EDANS

Hepatitis C virus Human cytomegalovirus Human immunodeficiency virus Acquired immunodeficiency syndrome US food and drug administration Direct acting antivirals Fragment based drug discovery High throughput screening Structure activity relationship Fluorescence (Förster) resonance energy transfer Nuclear magnetic resonance Surface plasmon resonance Non structural protein 3 4-((4-(Dimetylamino)phenyl)azo)benzoic acid 5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid

Introduction

Viruses are major threats to human health. Some viruses cause huge pan-demics and millions of people die each year from diseases caused by viruses. For example, more than 170 million people worldwide are infected with Hepatitis C virus (HCV), corresponding to 3% of the world’s total popula-tion. The virus is a small, rapidly evolving, single stranded RNA virus that causes a chronic infection in the liver which eventually leads to liver cirrho-sis and hepatocellular carcinoma [1]. Human cytomegalovirus (HCMV) is another virus with a severe outcome. This double stranded DNA virus has a slow replication cycle. HCMV infects between 50-85% of the world’s popu-lation and is able to establish a lifelong latency or persistence in infected individuals. In healthy people, the HCMV infection is often not associated with any diseases or symptoms. However, for individuals with a weakened immune system, like AIDS patients, organ transplant recipients or new-born babies, the infection can be life threatening [2, 3]. There is currently no vaccine available for either of these two viruses, but major progress has been made in the development of a HCMV vaccine [4]. For the rapidly replicating HCV, the development of an effective vaccine remains difficult. Anti HCV research has therefore instead been focused on the design and development of direct acting antiviral drugs (DAA). These drugs selectively target a protein involved in a crucial step in the viral life cycle, and have gained great success in the past decade. For HCMV, a num-ber of drugs, most targeting the DNA polymerase, have been launched on the market [5]. The HCMV protease is also considered a suitable drug target because of its crucial function in virion assembly. However, its flexible structure makes it is a challenging target and there is thus still no drug target-ing the protease available on the market. For treatment of chronic HCV, the NS3 protease is considered as one of the most attractive drug targets. Three DAAs targeting the NS3 protease have recently been approved by the US food and drug administration (FDA) for clinical use. Several other NS3 pro-tease inhibitors are currently being evaluated in clinical trials [6, 7]. Despite the success of several potent DAAs, the rapid emergence of resistance against the inhibitors limits their long term use. Drug resistant mutants have been observed for all clinical HCV drugs, and therefore there is an urgent need for new drugs with novel structures and mode of action.

11

The focus of the work presented in this thesis has been to improve the design of inhibitors for the proteases from HCV and HCMV. This will aid the fu-ture development of novel, more robust DAAs that are resilient to drug re-sistance and genetic variation. Both clinical drugs and novel inhibitors of the HCV NS3 protease have been evaluated using structure activity relationship (SAR) analysis and structure kinetic analysis (SKR) of different variants of the HCV protease, including different genotypes and drug resistant forms of the enzyme. For the HCMV protease, a fragment library has been screened against different variants of the enzyme in order to identify novel scaffolds for inhibitors.

Antiviral drug discovery Antiviral drug discovery is the process by which small molecules that inter-act with, and inhibit a target protein involved in a viral infection, are identi-fied and further developed into drugs for clinical use (Figure 1).

Figure 1. A simplified scheme of the drug discovery process.

Almost every protein that is involved in the viral life cycle, from cell entry to replication and release, can be considered as a potential drug target, although some proteins are more eligible as drug targets than others. Many viruses depend on both viral and host cell factors to be able to propagate in the host. However, to avoid undesirable side effects, the target should preferably be a viral protein that is essential for replication of the virus.

Target validation A drug target can either be a structural element of the virus particle, also known as the virion, or it could be a non-structural protein that is directly involved in the viral replication. Once a tentative target protein has been identified, it is vital to confirm that it is able to bind to a ligand that inter-feres with its function and that this interference eventually will eradicate the virus from the host. The drug target is considered to be validated when its role in the disease mechanism has been established and when it has been demonstrated that infection can be treated by inhibition of its function by a ligand.

Target identification and validation

Assay develop-

ment

Hit identifi-cation

Lead optimi-zation

Clinical trials

12

Assay development After validation, assays are developed and optimized for characterization of the target protein. Since the focus of this thesis is on proteases, the rest of this section will consider the drug target as if it was an enzyme. Hence, ki-netic assays that determine its basic enzymatic parameters are required. Dif-ferent model systems can be used, like parts of the replication machinery or individual enzymes. The enzyme can also be truncated for studies of specific parts or domains. However, the use of such truncated model proteins has drawbacks, since the deleted parts might influence the conformation and activity of the protein.

Hit identification The first step in the discovery of a new drug is to identify molecules, hits, which are able to interact with the target protein. There are several ap-proaches that can be used in this step. In rational drug design, the identifica-tion of a binding compound is based on the structure of the target protein. The design of these molecules has been guided by known features of the active site, the substrate and the catalytic mechanism. Another approach is to screen libraries of synthetic chemical compounds against the target. The compounds can either be quite complex and drug-like as in high throughput screening (HTS) or they can be small fragments, as in fragment based drug discovery (FBDD). In fragment based screening, the libraries are smaller than in HTS because the probability that a small frag-ment bind to the target is higher than for a more complex compound. By fragment screening, novel scaffolds with alternative binding sites or mode of action can be identified. Surface plasmon resonance (SPR) biosensor tech-nology has emerged as an alternative to X-ray crystallography and nuclear magnetic resonance (NMR) for identification of fragments, due to its sensi-tivity and ability to detect weak interactions. Interacting fragments are fur-ther validated by enzyme activity assays to confirm inhibitory properties.

Lead optimization Lead compounds are generated by optimizing hits with respect to potency, efficiency, stability, selectivity and pharmacokinetic properties. The chemi-cal modifications are guided by extensive analyses of SAR and SKR in combination with computational modeling. The potency of a drug is propor-tional to the affinity for the target protein and is often used to compare dif-ferent compounds. A potent drug is however not always the most efficient from a therapeutic point of view because the therapeutic effect can remain poor even if the drug is very potent. The selectivity is the ability of a drug to preferentially produce a desirable specific effect and it is an important pa-

13

rameter to predict in order to prevent severe side effects. The lead com-pounds undergo further extensive biological and pharmacological testing in different viral cell assays and test organisms. By examination of its pharma-cokinetic properties, explained by ADME (absorption, distribution, metabo-lism and excretion), its bioavailability can be optimized and undesirable side effects can be detected. When the lead compound has been developed into a candidate drug, it will enter clinical trials. Drug resistance is a great problem in the treatment of rapidly evolving virus-es. It is therefore important to integrate existing knowledge about the molec-ular basis behind drug resistance in an early stage in the drug discovery pro-cess.

Viral evolution Evolution is a natural part of the viral survival strategy. Some viruses, like HCV, can rapidly adapt to its human host for survival and continued trans-mission. This fitness driven genome evolution together with random se-quence drift has resulted in large genetic diversity among the virions of the Earth. Viruses also utilize its evolution to escape antiviral drug treatment. By its ability to develop drug resistance, the inserted drug may become futile and the virus can continue its replication in the host. The main focus of this thesis will be on the HCV protease, and therefore the discussion in the fol-lowing two sections is based on this virus.

Genetic variation of HCV

Based on genome sequence diversity, HCV has been partitioned into six major genotypes (1-6) that are further divided into several subtypes (a, b, c, etc). The genotypes differ from each other by 30% - 35% in the nucleotide sequences and the subtypes can differs by as much as 20% - 25% at the pro-tein level [8]. Genotypes 1 and 3 are distributed worldwide (Figure 2). Type 1a and 1b account for 60% of all global infections and predominates in Eu-rope, North and South America. Genotype 3 is most prevalent in Southern Asia, India and Australia but also occur in Europe and in the United States. Genotype 2 causes infections primarily in Western Africa while genotype 4 dominates in the Middle East and Egypt. Genotype 5 is mainly restricted to South Africa and genotype 6 is found in specific regions in Asia [8].

14

Figure 2. Global prevalence of HCV infections (WHO, 2008).

Drug resistance The rapid replication rate of the HCV (1012 new virions per day in an infect-ed individual) and the lack of proof reading by the viral RNA-dependent RNA polymerase, NS5B, lead to a high mutation rate and to a heterogenous population of virus variants in the infected individual, known as a qua-sispecies [9]. As a consequence, even before antiviral therapy is initiated, resistance associated mutations are already present at low copy numbers. Under the selective pressure of protease inhibitors, these resistant mutants in the quasispecies soon become dominant in the viral population, and fully developed drug resistance is a fact. During treatment with DAAs, the selective pressure from the drug favors fixation of specific mutations in the viral genome. Among these are amino acid substitutions in the inhibitor binding site of the target proteins. Drug resistance occurs when an amino acid substitution leads to a weakened affin-ity for the inhibitor without affecting the biological function of the target. Therefore, inhibitors that extent beyond the natural substrate binding site are highly prone to become ineffective due to drug resistance, since mutations outside the substrate binding pocket weaken only the affinity for the inhibi-tor without affecting the affinity for the natural substrate [10]. Drug resistance is a major challenge in antiviral drug discovery and large efforts are invested to circumvent this problem. Increasing knowledge about the structure of the target and modern sensitive biophysical technologies will hopefully soon result in the availability of, new more robust drugs that are less susceptible to drug resistance and more tolerable against genetic varia-tion.

1a, 1b, 2, 3

1a, 1b, 2b, 3a

1b

1, 2

1, 2, 3

3a, 1a, 1b5

2

1a, 1b

1, 2, 3

3, 1b

1, 23,6

6

4a

1, 4

4c, 4d, 1, 3

1a, 1b1b, 3a

1, 3 1b 1b

15

Viral proteases as drug targets A protease is an enzyme that catalyzes the hydrolysis of peptide bonds and is for example involved in the cleavage of other proteins. This reaction is often described as proteolysis. Viral proteases are key players in the viral replica-tion machinery and have therefore emerged as important drug targets. In general, proteases have well defined substrate binding sites and design of substrate analogous inhibitors has hence been a great success. When protease substrates or inhibitors are described, the Schechter-Berger nomenclature is often used [11]. Each residue in the substrate or inhibitor is numbered starting from the cleavage site, or scissile bond. On the N-terminal cleavage side of the scissile bond, the positions of the side chains are desig-nated P3, P2, P1 and on the C-terminal side P1’, P2’, P3’. The corresponding pockets, or subsites, in the protease are called S3, S2, S1, S1’, S2’, S3’ (Fig-ure 3). This nomenclature is used for peptidomimetic inhibitors and will be used throughout this thesis.

Figure 3. A substrate peptide bound in the active site of a protease. The positions of the residues in the substrate (P) are numbered relative to the cleavage site, the scis-sile bond, located between P1 and P1’. The corresponding subsites in the protease are denoted S. Nomenclature according to Schechter-Berger.

The HCV NS3 protease is responsible for processing of the polyprotein pre-cursor encoded by the single open reading frame in the viral genome. The HCMV protease processes the capsid assembly protein precursor and is hence essential for virion maturation. Both of these enzymes are classified as serine proteases because they have a nucleophilic serine in the active site. However, they are structurally very different. In this thesis, the main focus will be on the HCV protease, and therefore its replication, prevalence and pathology is described in more detail than for the HCMV protease.

S2 S1’ S3’

S3 S1 S2’

Protease

Substrate

Protease

N

O

N

H

R2

O

N

H

R1

O

N

H

R

N

H

R3 R

O

N

H

R

O

CH3

H

H3CP3

P2P1

P1'P2' P3'

The scissile bond

16

Hepatitis C virus NS3 protease The viral 9,6 kb RNA comprises a single open reading frame (ORF) that encodes a polyprotein precursor of 3010 amino acids. The N-terminal part of the ORF codes for three structural proteins (C, E1 and E2) that form the viral particle. The remaining part of the ORF codes for seven nonstructural pro-teins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) that are essential for viral function and replication. Upon replication, the polyprotein is processed by both cellular and viral proteases. The non-structural protein 3 (NS3) pro-tease is responsible for the proteolysis at the four cleavage sites between the non-structural proteins NS4A-NS5B that build up the replication machinery (Figure 4). NS4A is a 54 residue polypeptide expressed immediately down-stream NS3 in the viral polyprotein and the incorporation of its central part into the NS3 protease is essential for efficient proteolytic processing. In vi-vo, the N- -helix that anchors the NS3/4A complex on the endoplasmatic reuticulum where the viral replication takes place [12]. NS4B is a hydrophobic trans-membrane protein that is a key player in the formation of the membrane associated rep-lication complex, the membranous web [13]. NS5A is a phosphoprotein es-sential for RNA replication and particle assembly [14, 15], while NS5B is a RNA dependent RNA polymerase (RdRp) that catalyzes the synthesis of new viral RNA [16]. NS5B together with NS3 are considered to be the most important and well-studied targets for HCV drug discovery.

Figure 4. The HCV polyprotein. The NS3/4A protease is responsible for the proteolytic processing of the four junctions between NS3 and NS5B in the polyprotein precursor (black arrows). The other sites in the polyprotein are cleaved by host signal proteases (dashed ar-rows) and the NS2-3 protease (white arrow).

Another important function of the NS3 protease is cleavage and thus inacti-vation of two human cellular signal proteins involved in the innate immuno-logical response [17, 18]. Inhibition of NS3 may thus not only reduce viral replication in the host cell, but may also protect the innate immune response. The consensus sequence from P6 to P4’ in the natural substrates for the NS3 protease is Asp/Glu-X-X-X-X-Cys/Thr-Ser/Ala-X-X-X, where X is variable and the scissile bond is located between cysteine or threonine and serine or alanine [19].

Structuralproteins Non-Structural proteins

E1 E2 p7 NS2 NS3 4A NS4B NS5A NS5BCH2N- -COOH

17

Figure 5. Full length non-structural protein 3 of HCV. The active site of the prote-ase is located at the interface between the protease domain (pink) and the helicase domain (green). The catalytic triad, H57-D81-S139 is represented as sticks (tur-quoise) and the zink as a blue sphere. The central activating part of NS4A (yellow) is incorporated into the protease domain.

NS3 protease is a chymotrypsin like serine protease that is stabilized by a Zn2+ ion and its cofactor NS4A (Figure 5). A histidine (H57), an aspartic acid (D81) and a serine (S139) constitute the catalytic triad in the active site. The protease and the viral helicase constitute two separate domains of the full length NS3 protein. The function of the NS3 helicase/NTPase is to un-wind and separate double stranded RNA during viral replication. As both domains are functional on their own, truncated variants of either protease or helicase can be used as model systems in biochemical studies. However, in such a simplified model for the protease, the substrate binding site of the protease is shallow and relatively featureless with few interaction points for the binding substrate [20]. In contrast, the crystal structure of the full length protein reveals that the active site of the protease is located at the interface between the two domains, creating a closed and well-defined binding cleft [21]. The helicase can thus compensate for the lack of substrate interaction points in the protease [22, 23]. Additionally, studies have also shown that the two domains influence each other’s activity in the full length protein [24, 25]. It is still not clear how the NS3 protein can switch between its two func-tions, but it is believed that it might be regulated by allosteric modulations [26].

Zn2+

NS4A

Active site residues

18

Inhibitors against HCV NS3 protease Before the three dimensional structure of the full length HCV NS3 protein was solved, it was considered rather unlikely to find a direct acting protease inhibitor due to the shallowness of the protease active site. The standard of care for treatment of patients with chronic hepatitis C was thus restricted to combination therapy with the indirectly acting antiviral drugs pegylated in-terferon- [27]. However, resolution of the full length crystal structure of NS3 together with the discovery that the protease was inhibited by its own N-terminal cleavage product, represented an important starting point for the development of the first HCV NS3 inhibitors [28]. The hex-apeptide cleavage product was modified and optimized by rational drug de-sign. This led eventually to the discovery of BILN-2061 (ciluprevir) which was the first HCV NS3 protease inhibitor to enter clinical trials. By introduc-ing a macrocycle and a large bulky substituent on the P2 residue the stability of the peptide was increased and its flexibility decreased (Figure 6).

Figure 6. A hexapeptide derived from the cleavage product of the HCV NS3 prote-ase (left) was developed into BILN2061 (right), the first HCV NS3 protease inhibi-tor to enter clinical trials. The common P2 proline is marked in the figure.

BILN-2061 provided proof of concept for inhibitors with low nanomolar affinities, but was unfortunately later withdrawn due to toxicity in monkeys [29]. Today, three DAAs targeting the NS3 protease has been approved for clinical use in combination with interferon (IFN) and ribavirin and numerous other candidate drugs are currently being tested in clinical trials [6]. The compounds VX-950 (telaprevir) and SCH 503034 (boceprevir) were launched on the market in 2011 and are said to be “first-wave” HCV NS3 protease inhibitors. TMC435 (simeprevir) was launched in November 2013 and is said to be a “second-wave” protease inhibitor.

N

ONH

O

OHSH

O

NH

CH3H3CO

HN

H3C

CH3

O

NH

OH

OOHN

O

OH

H3C

O

O

N

ONH

NN

S

HN

O

OH

OO

NH

O

O

BILN 2061 Cil i

P2P2

19

Figure 7. Summary of the two strategies used for development of existing HCV NS3 protease inhibitors. In the rational approach, the inhibitors have been designed on the bases of the product and developed into macrocyclic and linear inhibitors. By fragment-based drug discovery, an allosteric compound has been identified and developed into a candidate drug.

Simeprevir has different structural features and several advantages over the “first-wave” compounds. It has a higher genetic barrier to resistance and better pharmacokinetic properties. Both the “first wave” and the “second wave” compounds are peptidomimetic transition state analogues of the natu-ral substrate.HCV NS3 inhibitors can be divided into linear compounds

20

(such as boceprevir and telaprevir) and macrocyclic compounds (such as ciluprevir and simeprevir) (Figure 7). They differ both in structural features and in interaction mechanisms. The macrocyclic compounds inhibit the pro-tease with a reversible non-covalent mechanism. In contrast, an electrophilic moiety introduced at the C-terminus of the linear compounds allows them to covalently but reversibly interact with the catalytic serine [30]. These elec-trophilic inhibitors are also called “mechanism based inhibitors”. The two “first-wave” compounds telaprevir and boceprevir are both linear inhibitors and the “second-wave” compound simeprevir is macrocyclic. ITMN-191 (danoprevir) is another macrocyclic inhibitor with a reversible non covalent interaction mechanism that is currently evaluated in clinical trials. Recently, a new class of inhibitors for HCV NS3 was developed by Astex Pharmaceuticals. A new allosteric binding site located at the interface be-tween the helicase and the protease domain of NS3 was identified by frag-ment based screening combined with X-ray crystallography. The binding fragments were further optimized and developed into a lead compound by structure guided design. The lead compound revealed that this specific and highly conserved site inhibited the protease activity via an allosteric mecha-nism. Compounds binding at this site also displayed a different resistance profile than inhibitors targeting the active site [26].

HCV protease inhibitor resistance Drug resistance has been observed for all peptidomimetic HCV protease inhibitors in Figure 8, both in in vitro studies using enzyme and replicon assays and in human clinical trials [31-33]. The most frequently observed amino acid substitution in or close to the active site affect A156, R155 and D168 (Figure 8). It is believed that the P2 moiety is the common denomina-tor for mutations in these residues.

Figure 8. The amino acids a) alanine (A), b) arginine (R) and c) aspartic acid (D) in positions 156, 155 and 168 respectively represented as yellow sticks and the active site residues as turquoise sticks.

a) A156 b) R155 c) D168

21

It has been shown that resistance interfering mutations are more likely to be occurring at sites where the inhibitor extends beyond the natural substrate binding site [10]. The binding of the inhibitor is hence weakened, while the affinity of the natural substrate is maintained. Consequently, an inhibitor that is designed to fit inside the natural substrate binding site is less sensitive to mutations. A common feature for all inhibitors depicted in Figure 7 is a pro-line in the P2 position, with a bulky substituent that protrudes from the sub-strate binding site and interacts closely with R155 and A156. Additionally, the P2 proline in combination with a P1 residue that is highly optimized for the wild type enzyme seems to be a major determinant for the lower potency against the resistant variants A156T and D168V [34].

Human cytomegalovirus protease Human cytomegalovirus (HCMV) protease is involved in the assembly of the viral capsid and consequently it has a crucial function in the release of mature infectious virion particles into the cell [35]. The protease exists in a monomer-dimer equilibrium (Figure 9), but it is only catalytically active as a dimer [36].

Figure 9. The dimer of human cytomegalovirus protease. The catalytic triads S132-H67-H157 in the two active sites is represented as green sticks.

The crystal structure of the protease reveals that it belongs to a novel class of serine proteases with unique fold and a catalytic triad consisting of a serine (S132) and two histidines (H63 and H157), instead of the typical serine-histidine-aspartic acid arrangement [37, 38]. The two active sites in the di-

22

mer are spatially separated from each other and do solely consist of residues of one particular monomer. However, mutations in the dimer interface have been shown to cause a significant reduction of the catalytic activity [39].

HCMV protease inhibitors There are currently no drugs on the market targeting the HCMV protease, although several inhibitors have been reported in the literature. Instead, the FDA-approved drugs for treatment of HCMV infection target the DNA pol-ymerase. Since the substrate binding pocket of the protease is relatively shal-low and structurally undefined, it is challenging to find a suitable active site binding inhibitor. An alternative approach could therefore be to develop inhibitors that target allosteric sites, potentially disrupting the dimer inter-face and thus affect protease functionality.

Evaluation of protease inhibitors For inhibition of a protease activity to occur, the inhibitor and the enzyme must encounter one another and form a binary complex by binding of the inhibitor to a specific site on the enzyme. This site could either be the active site, in which the enzyme carries out its catalysis on a specific substrate, or an alternative site, at which catalysis can be regulated allosterically. Differ-ent techniques can be used to study the interactions. With enzyme activity assays, the inhibitory potential can be obtained. With direct binding assays, like biosensor technology, information about how strong the inhibitor binds (affinity), how fast it binds to the target (association) and how fast or slow it come off (dissociation) can be provided. Some techniques, like X-ray crys-tallography, provide structural information about the interaction. It is an advantage to use several of these methods because they can complement and validate each other. The most common means to study inhibitor binding is by kinetic analysis of enzyme catalyzed reactions. This is an indirect method where a substrate is used as a reporter of the interaction. Another method is to study the interaction directly without the requirement of a substrate. In the next section, a qualitative model of enzyme reactions, referred to as Michae-lis Menten kinetics, is described.

Brief introduction to Michealis-Menten kinetics The steady state kinetics of all enzymatic reactions of Michaelis-Menten type is described by the following generic scheme:

(1)

23

E, S and P notify free enzyme, free substrate and free product, respectively, while ES is enzyme bound to substrate or product. The Michaelis-Menten parameter kcat/KM is the rate constant for association of free substrate to free enzyme multiplied by the probability that formation of the enzyme-substrate complex eventually leads to product formation and dissociation. kcat is the maximal turnover rate of the enzyme and KM is the substrate concentration at which the turnover rate of the enzyme is half maximal, i.e. kcat/2. The steady state rate of product formation for such enzymes, v0, is given by

(2) where kcat · [E0] = vmax. The general properties of Michaelis-Menten kinetics may be illustrated by the simplistic scheme:

(3) Here, k1 and k-1 are the rate constants for substrate association to free en-

zyme and substrate dissociation from substrate bound enzyme, respectively, while k2 is a compounded rate constant for the catalytic rate of S to P trans-formation and dissociation of P from the enzyme. In this simple case kcat = k2. After formation of the ES complex the probability of product formation and release rather than substrate release from ES is given by k2/(k2+k-1) and, hence,

Competitive inhibition Competitive enzyme inhibitors are “competitive” in the sense that binding of substrate and inhibitor molecules are mutually exclusive, as illustrated by the following scheme:

(4)

MM

cat

KSSv

KSSEk

v max00

21

21/kk

kkKk Mcat

24

Here, free enzyme, E, binds to free inhibitor, I, and forms the enzyme inhibi-tor complex, EI, with association rate constant kI. The enzyme inhibitor complex dissociates with rate constant k-I, and the enzyme catalyzed steady state rate of turnover of S to P is given by

(5)

where the inhibition (dissociation) constant, KI is given by KI = k-I / kI (6) It is seen that the presence of competitive inhibitors increases the KM-values of enzymatic reactions but leaves the kcat-values unaltered. In some cases, inhibitor binding to an enzyme is more complex than illus-trated in Scheme 4 above. When an inhibitor induces a conformational change of the enzyme or when the initial binding of the inhibitor to the en-zyme is followed by the formation of a reversible covalent complex, it could be illustrated by the following scheme:

(7) On the assumption that the second enzyme conformation only appears in the presence of inhibitor, the relation between inhibitor free and inhibitor bound enzyme can be written

(8)

where KI1 = k-I1 / kI1, KI2 = k-I2 / kI2 and

(9)

With the KI-value defined as an overall equilibrium (dissociation) constant according to Eqs. 8 and 9, the enzyme inhibition according to Eq. 5 remains valid.

)/1()/1(max0

0IMIM

cat

KIKSSv

KIKSSEk

v

1 2I

E IEI E I E I

K

21

2 1I

I II

KK K

K

25

Interaction analysis Enzyme-inhibitor interactions have been studied with two different assays in this thesis. An indirect activity based enzyme activity assay has been com-plemented with a direct binding assay using SPR biosensor technology. The two methods give different information about the inhibitors of interest and meet different needs in the process.

Activity based inhibition analysis In an enzyme activity assay, a substrate is used as a reporter of the enzyme activity and the initial reaction rate at steady state is measured. The interac-tion between enzyme and inhibitor is described by the equilibrium constant KI. Since proteases cleave a substrate into two products, Fluorescence (Förster) Resonance Energy Transfer (FRET) is a convenient method widely used for measuring proteolytic activity (Figure 10).

Figure 10. Principles for Fluorescence (Förster) Resonance Energy Transfer (FRET) assay for analysis of protease activity. A fluorescent donor (F) and a quenching acceptor (Q) are covalently attached to each side of the cleavage site, the scissile bond (SB), in a substrate peptide. When the substrate is cleaved by the protease the donor emits its energy as fluorescent light and product formation can be monitored.

The FRET protease substrate is a peptide with a fluorescent donor (EDANS, denoted F in figure 10) covalently attached to one end of the substrate and a quenching acceptor (DABCYL, denoted Q) at the other end. The peptide sequence of the synthetic substrate is derived from a natural cleavage site of the protease of interest. In the intact peptide, the energy from the excited donor chromophore is absorbed by the quenching group and dissipated as

F

F

Resonance energy transfer

SB Q

Protease cleavage

Q

26

heat. When the substrate has been cleaved, the distance between the donor and acceptor becomes very large and the energy from the excited donor group is no longer quenched. Instead the donor emits its energy as fluores-cence light. Hence, product formation can be conveniently monitored as fluorescence increase as a function of time. To determine the Michaelis-Menten parameters KM and kcat of a studied protease, the initial velocity is plotted as a function of substrate concentration and the parameters in the Michaelis-Menten equation are varied by non-linear regression for their best fit with the experimental data.

Selectivity and resistance analysis The activity of a vital enzyme in the presence as well as in the absence of inhibitors is vital for assessment of the risk of resistance development among pathogens. A convenient measure of comparison of a wild type (wt) with a mutated protein (mut) is the ratio between their steady state flows as de-scribed by Eq. 5 above at calibrated enzyme, substrate and inhibitor concen-tration levels:

where the “vitality index” V is given by [40]

It is seen that at large inhibitor and small substrate concentration the ad-vantage of the mutated in relation to the wild type enzyme is given by the vitality index V. It is also seen that varying the substrate and inhibitor con-centration may greatly affect the relative efficiency of mutated and wild enzyme; a feature that could be exploited in the struggle against resistance development.

Biosensor analyzed enzyme inhibitor binding In a direct binding assay, the interaction between two molecules is followed in real time without the use of a labeled substrate to monitor the activity. The equilibrium dissociation constant (KI) can thus be resolved into its individual rate constants. This type of data hence enables detailed characterization of the kinetic and mechanistic features of the interaction, like for example con-formational changes or complex mechanistic steps. SPR biosensor based technology is a highly sensitive method that allows detection of weak inter-actions. Therefore it has emerged as an alternative to NMR and X-ray crys-tallography in fragment based drug discovery.

0 0

//

/

wt wt wtI M Imut wt

mut mut mutI M I

S K K K Iv v V

S K K K I

/

/

mut mut mutcat M I

wt wt wtcat M I

k K KV

k K K

27

SPR is an optical phenomenon that occurs at the interface of two materials with different refractive index under total internal reflection. The SPR bio-sensor is composed of a glass layer coated with a thin gold layer. The gold layer is covered with a dextran matrix to which the first interaction partner, like the proteases to be studied here, can be covalently immobilized by dif-ferent chemical coupling methods. The second interaction partner, the ana-lyte, which in this work is the drug candidates, is injected over the sensor surface. The interaction between the two molecules results in a change in refractive index at the surface which is proportional to the mass change. This change is detected as a function of time and results in a sensorgram (Figure 11)

Figure 11. A simulated sensorgram representing a typical output from an experiment using a surface plasmon resonance biosensor. After an initial baseline has been es-tablished, the analytes are injected and binding between the two molecules is detect-ed in response units as a function of time. When the injection stops the analyte dis-sociates from the surface and the signal returns to base line.

Inhibition and interaction data and their relevance for drug design Interaction studies can be used at different stages in the drug discovery pro-cess. In the initial stage of the process, it may be sufficient to determine if a compound binds to its target or not. Later in the process it becomes more relevant to characterize the interaction in more detail. From a drug discovery point of view, an inhibitor with a fast association to its target and a slow dissociation, resulting in a high affinity is of advantage. In this thesis, the enzyme activity based inhibition assay has been used to study the structure activity relationship (SAR) of inhibitors in order to identi-fy features in the scaffold that may be the key determinants for emergence of resistance. It has also been used to validate interacting fragments identified by SPR biosensor technology. SPR biosensor technology has been used to obtain detailed information about the interactions between HCV NS3 prote-ase variants and structurally different inhibitors and for fragment screening.

28

Present investigation

Aim The aim of the present work was to develop new strategies for identification and optimization of novel antiviral drugs with improved potency, selectivity and resistance profiles. The specific objectives were to:

Identify residues in novel Hepatitis C virus NS3 protease inhibitors that are the common denominators for observed potency loss against the major resistant variants.

Provide a strategy for quantifying selectivity and resistance of HCV NS3 protease inhibitors

Identify novel chemical starting points for the design of HCMV in-hibitors

Production of proteases To fulfill these aims, the initial production of high quality proteins was of importance.

HCV protease variants In paper I, the wild type (wt) and the resistant variants A156T and D168V of the full length HCV NS3 protease from genotype 1a and in paper II and III, full length NS3 from HCV genotypes 1a, 1b and 3a, truncated NS3 harbour-ing only the protease domain and full length NS3 co-expressed with full length NS4A from genotype 1a and 1b were successfully expressed and puri-fied for the most part as previously described [41], but with some modifica-tions. Briefly, the proteins were expressed in E. coli and the cleared lysate was subjected to by immobilized metal ion chromatography (IMAC) of the. After desalting of the eluate, the full length NS3 proteins were further puri-fied with poly uracil (polyU) sepharose chromatography while the truncated

29

variants of NS3 were stored for further use. A synthetic peptide correspond-ing to the central part of the NS4A protein was used as NS4A cofactor.

HCMV protease variants In paper IV, six variants of the HCMV protease were expressed in E. coli and purified under denaturing conditions in urea with anion exchange and size exclusion chromatography. The variants were subsequently refolded by removal of urea with dialysis. The functionality of the refolded proteins was confirmed by activity measurements.

Interaction studies Two different approaches based on different molecular principles were used to study the interactions between enzymes and inhibitors in this work. In papers I and II, the interactions between inhibitors and the HCV NS3 pro-tease were studied with an enzyme activity assay, which is based on FRET. In paper III, SPR biosensor technology was used to study the direct binding between HCV NS3 protease variants and structurally variable inhibitors. In paper IV SPR was used for screening of fragments against HCMV protease variants.

Structure-activity analysis of HCV NS3 inhibitors (Paper I) A common structural feature of HCV NS3 protease inhibitors that are cur-rently or have been evaluated in clinical trials is the presence of a proline at the P2 position. It has been suggested that the residue in the P2 position has a great impact on the susceptibility to drug resistance [10, 42]. A proline in this position has been shown to be a major determinant for the reduced effi-cacy against resistant mutants and that alternative moieties are required for maintained potency. In previous studies, a phenylglycine in the P2 position has been suggested as a suitable P2 residue due to its ability -stacking interaction with the catalytic His57. Compounds containing this P2 moiety have also shown retained potency against the resistant mutations R155Q, A156T and D168V [43, 44]. Additionally, the P1 residue was shown to have a major influence on the inhibitory potency against the substituted enzyme variants. In order to explore these residues more thoroughly, and to determine their role in resistance development, a structure-activity relation-ship (SAR) study was performed with a set of P2 proline or phenylglycine tripeptide inhibitors with different P3 (R) and P1-P1’ (R1)-groups. We inves-

30

tigated how the inhibitory potential was influenced by the resistant substitu-tions A156T and D168V, with the vitality value being used as a measure of the biochemical fitness of the resistant enzyme variant in the presence of inhibitor. As can be seen in Table 1 for the P2 proline series there is a clear correlation between the inhibition effect on the wild type enzyme and the susceptibility to mutations. The more optimized the P1-P1’ residue was for the wild type target (compound 1-3, Table 1) the greater became the loss in inhibition potency when the structure of the target changed. Hence, the combination of a P2 proline with a highly optimized P1residue seems to be the key determi-nant for resistance.

Table 1. Ki and V values for P2 proline-based inhibitors. (SD, standard deviation)

No such correlation was seen for the phenylglycine based counterparts (compounds 5 and 6, Table 2) as the potency towards the wild type was re-tained for the resistant mutants. Overall, the inhibition potency was lower for the phenylglycine-based series than for the proline-based series. This can be attributed to the fact that the P1-P1’ blocks are optimized for the P2 proline scaffold and not for P2 phenylglycine. However, compound 7 contains a P1 residue that is more suitable for phenylglycine than for proline and for which

WT A156T D168V

Cpd R R1 KI (nM) ± SD KI (nM) ± SD V KI (nM) ± SD V

1 H

230 ± 40 69 ± 23 0.4 1350 ± 500 8.6

2 H 55 ± 7.0 2300 ± 380 58.4 6100 ± 1500 115

3 Me 0.34 ± 0.05 66 ± 10 271 200 ± 28 610

4 Me 830 ± 110 830 ± 150 1.4 780 ± 81 1.0

31

the potency was almost three times higher than that of its proline counterpart (compound 4). This example indicates that the P2 phenylglycine based in-hibitors may have a different binding profile than compounds with P2 pro-line.

Table 2. Ki and V values for P2 phenylglycine-based inhibitors. (SD, standard deviation)

Molecular modeling was performed to rationalize the biochemical results (as described in paper [34]). Inhibitors 1, 3, 5 and 6 were docked in the active sites of the wild type and the A156T variant, providing insights regarding what causes the experimental differences in inhibitory potency. The P2 phe-nylglycine-based inhibitors were shown to retain their wild type confor-mation in the substituted variant while the binding conformation for the P2 proline based inhibitors were conspicuously different between the two vari-ants.

Conclusion Optimization of the P1-P1’ blocks for P2 phenylglycine inhibitors may be a valid strategy to obtain novel inhibitors with retained potency on resistant mutations as well as the wild type.

WT A156T D168V Cpd R R1 KI (nM) ± SD KI (nM) ± SD V KI (nM) ± SD V

5 H

280 ± 20 630 ± 120 3.1 710 ± 86 2.6

6 Me 230 ± 50 300 ± 54 1.8 330 ± 27 1.5

7 Me 310 ± 60 230 ± 36 1.0 330 ± 57 1.1

32

Evaluation of HCV NS3 inhibitors (paper II) In previous described paper, full length HCV NS3 variants from genotype 1a was analyzed. In order to enable the design of inhibitors with lower suscep-tibility to resistance and genetic variation, suitable model systems and exper-imental conditions needed to be identified. We therefore produced a set of NS3 variants from the prevalent strains 1a, 1b and 3a and subsequently eval-uated their sensitivity to clinically relevant inhibitors. Full length NS3 from genotypes 1a, 1b and 3a, harbouring both the protease and helicase domains were analysed in the study. Truncated NS3, constituting only of the protease domain and full length NS3 co-expressed with full length NS4A from geno-types 1a and 1b were also analysed in order to clarify the role of the helicase and the cofactor for the interaction. In paper II we evaluated and compared the inhibition potential, KI, of selected inhibitors against the different prote-ase variants, including the drug resistant variant R155K of genotype 1a. The compounds used for this study (VX-950, N-1725, BILN-2061, SCH 503034 and ITMN-191) all bind to the active site but differ in molecular structure, inhibition potency and interaction mechanism (see Figure 7 above). The catalytic properties of the different enzyme variants were shown to dif-fer significantly in the buffer previously used for studies of full length NS3 from genotype 1a (paper I). To compare the inhibition of each variant by the compounds in an appropriate way, the buffer conditions (detergent, glycerol and salt concentration) were for each variant adjusted for maximal enzyme activity at saturating substrate concentration (kcat-value). In this way we monitored inhibition of each variant under the condition of its best perfor-mance in the absence of the drug. Another way of comparison would be to design a reference buffer system mimicking that in the living cell and use this to compare all the variants with respect to catalytic efficiency and drug sensitivity. This is rendered difficult due to lack of knowledge of the in vivo condition, but the approach is principally attractive. However, in this study we were rather interested in the specific properties of the inhibitors, like their potencies, selectivity and mechanisms. In this experimental design, the en-zyme variants were given the best possible conditions to resist the inhibitors. By comparing the catalytic properties of the different genotypes under vary-ing buffer conditions, important information about their dependencies on the helicase domain and the NS4A cofactor was also obtained. For example, the truncated protease from genotype 1a had similar catalytic properties as the full-length NS3 protein, once optimized conditions had been identified. In contrast, there was a major difference in the catalytic properties of the full-length and truncated versions of the genotype 1b enzyme, indicating that the proteolytic activity depends more on the helicase domain for genotype 1b than 1a. The KM values for the full length variants from the three genotypes, including the resistant mutation R155K from genotype 1a were very similar.

33

This is explained by the fact that the overall conformation of the substrate binding site is conserved among the variants. The catalytic efficiency, kcat/Km, was highest in the optimized buffer for most of the variants. This is expected, since the buffer was in each case optimized for maximal kcat. What is more interesting is that the optimal buffers also tended to lower the KM values of the interaction. These results highlight the importance of using suitable model systems and optimized experimental conditions for the subsequent evaluation of inhibition. The inhibitory potential, KI, was shown to depend strongly on the type of protein variant as well as on buffer condition. All inhibitors were generally more potent against genotype 1a and 1b than genotype 3a. This is expected since these inhibitors had been developed for inhibition of genotype 1 prote-ase. For example, the KI value of BILN 2061 for the full length variant from genotype 3a was almost 600-fold higher than that from genotype 1a. This difference can be explained by the genotype 3-residue Gln168 in the inhibi-tor binding site. This has been identified as the major determinant for the loss of affinity for BILN 2061 for the genotype 3a protease [45]. In genotype 1a and 1b an aspartic acid is instead in this position. The large variation in KI-value among the variants indicates that the binding of inhibitor is more sensitive to natural genetic variation than the binding of substrate peptide.

Figure 12. Vitality values for the four inhibitors. Full length NS3 from genotype 1a was used as a reference.

To compensate for the changes in catalytic properties of the enzyme vari-ants, vitality values were used to compare the inhibitors efficiency on the variants. Hence, the differences in resistance and selectivity of the variants could be evaluated. The kinetic data for full length NS3 from genotype 1a

34

measured under the optimized experimental conditions was set a reference. The vitality values illustrated in figure 12 show that all inhibitors are most potent on the genotype 1a enzyme and least efficient against genotype 3a and the resistant mutant R155K (vitality value>>1). The inhibitor with broadest selectivity and best resistance profile was VX-950.

Conclusions This study provides a procedure for biochemical evaluation and comparison of inhibitors against different clinically relevant genotype and resistant vari-ants of the HCV NS3 protease. The analysis established that the linear com-pound VX-950 had the broadest selectivity and the lowest propensity for resistance compared to the other compounds.

Detailed characterization of NS3 inhibitors (Paper III) The inhibitors studied in paper II were in this paper characterized in more detail using SPR-biosensor technology. By resolving the interaction into its individual rate constants, the kinetic and mechanistic features of the inhibi-tion could be obtained. The protein variants were immobilized to the SPR biosensor surface and the inhibitors were subsequently injected in concentration series. To identify the interaction mechanism and estimate the rate constant and affinities, different models describing a 1:1 interaction, an interaction with a heterogenous lig-and and a two state interaction were fitted to the sensorgrams by non-linear regression. A two-state model was chosen since it gave the best fit for all interactions. This model is representative for both an induced fit mechanism in which binding of the inhibitor is followed by a conformational change of the enzyme, and mechanism-based inhibition for which binding of the in-hibitor is followed by the reversible formation of a covalent complex. Here only the full length variants are analyzed. For a broader analysis involving also the protease domain variants and the influence by the cofactor NS4A on the interaction, see the published paper. In order to visualize the results, the rate constants for the first (k1 and k-1) and second interaction steps (k2 and k-2) were plotted in interaction kinetic plots (Figure 13). (These are denoted in a different way in scheme 7 but represent the same constants). This enabled the comparison of inhibitors and the char-acterization of advantageous features in the interaction kinetics. The com-pounds with highest affinity for the first step (KD) were positioned in the top left hand corner in the k1 vs k-1 plot. The compounds with this position in the k2 vs k-2 plot had a second step that contributes significantly to the overall affinity (KD*). The overall affinity is the same as KI defined in equation 9 above. However to distinguish from the dissociation equilibrium constants

35

obtained from the two different assays, the overall affinity is from the SPR biosensor is here denoted KD*. The quote KD/KD* was used as a measure of the impact by the second step to the overall interaction.

Figure 13. Interaction kinetic plots for a) k1 vs. k-1, and b) k2 vs. k-2, for the interac-tions between the inhibitors and the full length NS3 variants from genotype (gt) 1a, 1b and 3a, assuming a 2-step mechanism. N-1725; BILN-2061; ITMN191; -×VX-950; SCH-503034.

The macrocyclic compounds (BILN-2061 and ITMN-191) had faster associ-ation and slower dissociation than the linear compounds, indicating that ri-gidifying the compounds may be of advantage from an interaction kinetic perspective. BILN 2061 had high affinities for all genotypes, but the interac-tion kinetics were relatively genotype dependent. This can be due both to the macrocyclic structure that makes the compound less flexible in its interac-tion with the protease and to the fact that the compound is optimized for genotype 1. For genotype 3a the second step was shown to have a greater impact on the interaction than for 1a and 1b. This could be seen both in the k2 vs k-2 plot as a shift to the upper left corner and by the high KD/KD* (see Table 3).

1E+3

1E+4

1E+5

1E+6

1E+7

1E+8

1E-3 1E-2 1E-1 1E+0

k 1(M

-1s-1

)

k-1 (s-1)

1E+3

1E+4

1E+5

1E+6

1E+7

1E+8

1E-3 1E-2 1E-1 1E+0

k 1(M

-1s-1

)

k-1 (s-1)

1E+3

1E+4

1E+5

1E+6

1E+7

1E+8

1E-3 1E-2 1E-1 1E+0

k 1(M

-1s-1

)

k-1 (s-1)

1E-7

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E-6 1E-5 1E-4 1E-3 1E-2

k 2(s

-1)

k-2 (s-1)

1E-7

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E-6 1E-5 1E-4 1E-3 1E-2

k 2(s

-1)

k-2 (s-1)

1E-7

1E-6

1E-5

1E-4

1E-3

1E-2

1E-1

1E-6 1E-5 1E-4 1E-3 1E-2

k 2(s

-1)

k-2 (s-1)

NS3 gt 1a

NS3 gt 3a

NS3 gt 1b

36

Table 3. Equilibrium constants for the interactions between studied inhibitors and full length NS3variants from genotype 1a, 1b and 3a. The KD* is the overall affinity and KD=k-1/k1. The KI values are obtained from paper II.

This feature might be explained by a different rearrangement of the active site in genotype 3a that was not seen for 1a and 1b. ITMN-191 seems to be structurally more flexible compared to BILN-2061, showing almost equal affinities for all three genotypes. Although it was determined to be the least genotype dependent compound, the interactions between ITMN-191 and the 1a and 3a variants appear to be more influenced by the second step than the 1b variant. This could be due to a different binding mode for the genotype 1b variant, or to the experimental challenges encountered with this assay. This is seen by the great deviation of the KD* values from the KI-values for the same interactions obtained from the enzyme inhibition assay. Since the equi-librium of the interaction is the same, the KD* values are supposed to corre-late with the KI-values. For this interaction, the KI was 500 and 700 fold higher for 1b and 1a respectively compared to 3a. For comparison, the KD* was 4 times higher for 3a than for 1a and 3 times lower for 3a than for 1b.

NS3fl1a NS3fl

1b NS3fl3a

N-1725 KD (nM) 3900 7200 1400KD* (nM) 200 5600 870KI (nM) 3.2 1.24 39.1KD/KD* 20 1 2

BILN-2061 KD (nM) 3.0 220 3300KD* (nM) 1.3 67 160KI (nM) 0.05 0.11 29.2KD/KD* 2.3 3 21

ITMN-191 KD (nM) 2.5 1.6 21KD* (nM) 0.1 1.6 0.5KI (nM) 0.046 0.06 32.9KD/KD* 19 1 41

VX-950 KD (nM) 2000 8.6 66000KD* (nM) 340.0 8.5 1000KI (nM) 20.2 28.1 102KD/KD* 5.9 1.0 66

SCH-503034 KD (nM) nd 16000 1700KD* (nM) nd 90 3.9KI (nM) nd nd ndKD/KD* nd 178 436

37

This indicates that the assay needs to be optimized further before to ultimate conclusions can be drawn.

For genotype 1b, drug affinities were generally not significantly influ-enced by the second step of the interaction. The only exception is SCH 503034 which is like VX-950 a mechanism based inhibitor. The formation of a covalent adduct between the electrophilic group of VX 950 and the ac-tive site serine was more clearly seen for 1a and 3a because the second step had a greater impact on the interaction for these genotypes. For SCH 503034, this feature was clearer for 1b and 3a since the KD/KD* was very large. Unfortunately, no data for genotype 1a with SCH 503034 could be obtained due to the low immobilization levels of the protein and the low molecular weight of the compound.

For the prototypic N-terminal product peptide analogue N-1725, the affin-ities for all genotypes were relatively weak, as all points were positioned in the right lower corner in the k1 vs k-1 plot. The affinity for genotype 1b was lowest with a KD* of 5600nM. In contrast regarding the equilibrium constant from the inhibition measurements, the affinity for 1b is the highest for the three genotypes.

Conclusions This study highlights the importance of resolving interaction kinetics into their individual constants when inhibitors are evaluated. Hence, detailed information about specific features of the interaction could be revealed that could guide the design of novel and more efficient drugs that are tolerant against genetic variation and resistance. The analysis and comparison of three different genotypes (1a, 1b and 3a) revealed that there are clear varia-tions in the kinetic and mechanistic features of the interactions between the genotypes. The macrocyclic compound ITMN-191 that is currently in clini-cal trials, was shown to have faster association and slower dissociation kinet-ics for the first interaction step for all genotypes compared to the approved mechanism based compounds VX-950 and SCH 503034. It is worth men-tioning that ITMN-191 had the highest overall affinity not only for genotype 1a and 1b, but also for 3a, indicating that its structural features may be bene-ficial for a broad spectrum drug. The most recently launched HCV NS3 in-hibitor, Simeprevir (TMC 435), is a macrocyclic compound that resembles the ITMN-191 compound both in structure and mechanism of action. This feature might therefore represent the next generation of broad spectrum drugs. However, for proper validation of the results and before to decisive conclusions can be drawn from the structure-kinetics relationship data, com-parisons with the equilibrium constants obtained from an indirect enzyme activity-based assay is valuable. The compounds analyzed in this study are optimized for genotype 1, and therefore the affinities ought to be higher for this genotype than for genotype 3. This correlation is clearly seen for the KI-

38

values obtained from paper II, but not for the KD* values determined here. This indicates that there is still room for improvement in the experimental setup of this sensitive and powerful assay. This information revealed advan-tageous structural features of the compounds that could guide the design of future HCV NS3 protease drugs.

Fragment-based discovery of HCMV protease inhibitors (paper IV) An SPR biosensor based approach was used to identify novel scaffolds for inhibitors against HCMV protease. A fragment library was screened against a set of protease variants with different amino acid substitutions in the active site. This panel was designed in order to identify where in the active site a certain fragment bound. To validate the identified binders, the screening was complemented by enzyme activity based inhibition analysis. Six active site variants of HCMV protease were successfully cloned, ex-pressed, purified and characterized (Figure 14).

Figure 14. The active site of HCMV protease. The location of the catalytic residues is indicated by the blue surface and the substituted amino acids by a red surface. A covalent inhibitor bound to the active site is shown as a stick model with green car-bon atoms.

The substitutions gave rise to structural differences in the active site that affected the catalytic properties of the enzyme (Table 4).

39

Table 4. Kinetic parameters of the HCMV variants

When the small glycine (G) in position 164 was substituted to the large and hydrophobic phenyl alanine (F), the enzyme activity was abolished. The same inactivation was observed for the S134F substitution. This could either be due to a steric clash between the substrate and the large bulky phenyl alanine, or to incorrect folding of the mutant enzyme. The substitution of isoleucine at position 231 to phenyl alanine (I231F) gave rise to a 20-fold increase in KM compared to the wild type enzyme, while the kcat was unaf-fected. This residue is positioned somewhat distant from the active site but close enough to influence the affinity of the substrate to its binding site. Sub-stitution of leucine 133 to phenyl alanine however increased the kcat more than 50 times, while KM was unaffected. In general, KM was not dramatically changed. This suggests that the binding of the substrate might not be highly dependent on the exact conformation of the active site. After an initial screen of 930 fragments at a single concentra-tion, 160 potential hits were selected by applying different criteria. The sec-ond screen involved parallel analyses of the selected compounds with both SPR-based interaction and enzyme inhibition assays. The fragments were screened at four concentrations against three of the enzyme variants together with the wild type. These enzyme variants were selected on the basis that they showed the largest differences in their interaction with fragments com-pared to wild type in the first screen. However, in the second screen, there were no significant differences in how the 160 substances interacted with the four enzyme variants. This can be an indication that the active site is not the binding site for any of the identified hits or that it is structurally flexible and the low affinity interaction of the fragments is not different for the different variants. Consequently, the subsequent screening was performed without the mutant variants. 21 fragments selected in the second screen were further and more rigorously analyzed both with SPR biosensor and inhibition assay. Two compounds were validated by both methods. The obtained affinities

Enzyme Subsite

location

kcat

(s-1)

Km

(μM)

kcat/Km

(s-1μM-1)

wild type 0.108 15 0.0074

K237A S2’ 0.077 50 0.0016

I231F S2’ 0.111 328 0.0003

N62A S1’ 0.222 19 0.0120

G164F S1’ Inactive - -

L133F S2 0.002 21 0.0001

S134F S2 Inactive - -

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from the SPR assay were relatively low, and the KI values, determined from the enzyme activity based assay, were 527 and 177 μM respectively. To make sure that the compounds truly inhibited the protease and not simply interfered with the FRET signals, a non-fluorescent method was developed. In this assay, the amount of product formed by the enzyme was determined by HPLC and detected by UV. This experiment showed that actually only one compound had inhibitory properties and were hence further analyzed. Its structure suggested however that it might be redox active (Figure 15).

Figure 15. Structure of the identified hit.

In order to ascertain that the protease was not inhibited by an oxidative mechanism, the reducing agent dithiothreitol (DTT) was included in the assay buffer. The activity of the protease was consequently enhanced, sug-gesting that the enzyme already had been partly oxidized. But as suspected, the promising fragment did not inhibit the activity, indicating it indeed acted as a redox catalyst for sulfide oxidation.

Conclusions The flexible structure of the HCMV protease and its shallow substrate bind-ing site makes it a challenging drug target. There were several hits that inter-acted well with the protease, but they were shown to not inhibit its catalytic activity. They were therefore concluded not to interact with either the active site or an allosteric site. This unspecific binding could be explained by the high flexibility of the protein or the fact that a large amount of the protein was improperly folded protein. Lipophilic fragments could in that case bind to lipophilic residues in the target that, in a correct folded protein, otherwise would not be exposed. The lack of positive hits could also be due to the fact that the used fragment library was not diverse enough. This challenging tar-get might require compounds belonging to another chemical space that this library did not cover. Hence, other, e. g. natural extract-based libraries could be an attractive alternative.

OH

HN

H3C

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Conclusions and future perspectives

Conclusions The work in this thesis provides novel procedures for identification and evaluation of protease inhibitors. It highlights the importance of choosing suitable model systems and experimental conditions in the evaluation of viral protease inhibitors. It also emphasizes the importance of combining different techniques for characterization as well as for validation. The results provide important structural features that could guide future broad spectrum anti-HCV drugs. Fragments were also identified that may serve as starting points for the discovery of effective inhibitors against the HCMV protease.

Future perspectives There are several aspects of this work that I consider as interesting for fur-ther exploration:

With both enzyme activity measurements and biosensor analysis fur-ther explore allosteric binders and compare them with active site binding inhibitors.

Expand the panel of HCV NS3 protease variants used in this thesis with more resistant variants

Establishing a biosensor assay where the full length NS4A is linked to a reconstituted lipid membrane. By subsequent analysis of the in-teractions with other replication proteins as well as with inhibitors in this novel model system, the proper mechanism and regulation of NS3 protease can be better understood and new aspects of im-portance for HCV drug discovery can be revealed.

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Svensk sammanfattning

Utveckling av läkemedel mot virala sjukdomar Många virus orsakar pandemier världen över och vissa virusinfektioner är svårare att bekämpa än andra. Virus som snabbt förökar sig i den infekterade individen har en stor benägenhet att utveckla resistens mot insatt läkemedel. Därför finns det ett stort behov av nya läkemedel på marknaden med förbätt-rade egenskaper, som högre tolerans mot resistens och genetisk variation. Proteaser är enzymer som klyver andra proteiner och har ofta mycket viktiga funktioner i de flesta biologiska processer. Hos många virus spelar proteaser ofta en betydande roll i deras fortplantning. När man vill bekämpa ett virus brukar därför proteaser anses som attraktiva läkemedelsmål. Lyckas slå ut funktionen av ett proteas som är livsviktigt för virusets fortsatta överlevnad är chanserna goda att virusinfektionen kan bekämpas. Funktionen av ett pro-teas kan hämmas om en molekyl som liknar det protein som proteaset van-ligtvis klyver, binder till proteasets så kallade aktiva säte utan att själv kly-vas. Den molekylen blockerar således bara proteasets funktion, eller aktivi-tet, och kallas inhibitor. En inhibitor kan också reglera ett proteas aktivitet genom att binda till andra ställen än det aktiva sätet, den kallas då allosterisk bindare. Då evolution av resistens är ett stort hinder för behandling av många virusin-fektioner är det viktigt att ta det i beaktning i ett tidigt skede i läkemedelsut-vecklingsprocessen.

Min forskning Det övergripande målet med min forskning har varit att studera interaktion-erna mellan inhibitorer och de virala proteaserna från Hepatit C virus (HCV) och humant cytomegalovirus (HCMV) med syfte att bidra till utvecklingen av nya mer robusta läkemedel som är mindre känsliga mot resistens och genetisk variation. Olika tekniker har användas för dessa interaktionsstudier som i kombination har gett djup viktig kunskap om funktionen av dessa pro-teas och hur framtidens läkemedel mot dessa kan designas. Genom att mäta proteasets aktivitet i närvaro av inhibitorn kan man bestämma i vilken ut-sträckning inhibitorn hämmar proteaset och kan på så sätt bestämma förhål-landet mellan inhibitorernas strukturer och deras förmåga att blockera pro-

43

teasets aktivitet. Med denna information kan man således guida optimering-en av nya mer effektiva inhibitor. Med biosensorteknik kan man följa inter-aktionen i realtid och således få information om hur hårt inhibitorn binder (affinitet), hur snabbt den binder till (association) och hur snabbt den släpper från (dissociation) proteaset. Det ger en detaljerad vetskap om interaktionens mekaniska processer, något som är mycket värdefullt vid utvecklingen av nya läkemedel. Med biosensorteknik kan man även på ett effektivt sätt sålla ett stort bibliotek (screening) av många små molekylfragment för att kunna identifiera interaktioner. På detta sätt kan helt nya strukturer identifieras som kan byggas ut och antingen hämma proteaset från dess aktiva säte, eller al-losteriskt.

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45

Acknowledgments

During my years at the department of Chemistry-BMC, I have had the privi-lege to be surrounded by so many nice people that have encouraged, helped and supported me. Without you, this work had been impossible. First and foremost I would like to thank my supervisor Helena Danielson, for accepting me as a PhD-student. You have been inspiring, challenging and encouraging. My co-supervisor Micke Widersten, for giving me feedback on my research over the years. Gun Stenberg, for sharing your invaluable knowledge in bio-chemistry with me. Gunnar Johansson, for being a brilliant teacher. Doreen, for reading and giving me valuable comments on this thesis, thank you also for your hospitality and for providing us with delicious homemade cookies and cakes every now and then. Francoise, for all our interesting discussions about pedagogics and teaching. The medicinal chemists Anja Sandstöm, Eva Åkerblom, Pernilla Örtqvist, Anna-Karin Belfrage, Anna Lampa, Johan Gising and Gunnar Lindeberg for a fruitful cooperation and for interesting discussions about organic chemis-try. Especially I would like to thank Eva for input on this thesis and to Anna-Karin for help with structures. Bosse, for all the help with my computers.

Present members of the HD group and colleagues at the third floor: Christian Seeger, sharing office with you this last year has been a true privi-lege. You have introduced me to the magic world of LaTeX, showed me how to make nice images in Pymol and backed up my computer files. You have also been a great ski instructor (although you spent most of the time looking for me in the forest). Without your help, encourage and trust, I don’t think this thesis ever had been finalized. Eldar, you have a crazy (almost as bad as mine) sense of humor and the admirable ability to solve all the prob-lems you encounter, and mine as well, especially those I used to have with the 10-ml pipet. Helena N, for really understanding the phrase “This morn-ing was a little bit chaotic”. Sara Solbak, thanks to your incredible negotia-tion skills, we won the battle against the pipet company.

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Erika, for every morning knocking on my door with your sweet sunshine smile and telling me that everything will be ok. You are a great source of energy, a talented teacher and a kick-ass spinning instructor. Marcus, for being a skillful glögg maker. Dirk, for bringing new energy to the lab. Anna T, for being a super organizer and for having kakboden-a life saver during the writing of this thesis. Ylva, for keeping me company late nights at BMC. Former members of the HD group: Sofia, we started this journey together and you have been a wonderful friend ever since. Thank you for sharing the NS3 project with me and for all the support over these years. Tony, although there are still german-speaking SPR experts in the lab, it has been disturbingly empty here since you left. Johan W, only a talented engineer knows where the mysterious O-rings should be attached in the celldisruptor, and you proved that you are. Malin, with your endless source of energy and enthusiasm, you are inspiring. Göran, for in-troducing me to the NS3 project. Thomas, for all the help with my research projects during the years. Matthis, with interesting discussions about SPR, the föräldrafikas at daycare got so much more exciting. Ikram, for bringing genotype 3a to the lab all the way from Pakistan and for cooking nice food to us. Several undergraduate students I’ve had the opportunity to supervise during the years have been a great help. Especially I would like to thank Benjamin for being accurate, curious and extremely efficient and Inga, for all the work with the HCMV project. All the nice organic and biochemists at the fourth floor: Sara Norrehed, you are a true talent, a great friend and fabulous party-partner. Soon, there will be confetti rain and balloons all over the place. Jo-hanna J, thank you for helping me organize my life every now and then and for making me aware of every time I forgot my mascara. When this is over, the therapy will be open for you again. Johan V, you are a charming fairy. Cissi, for (almost) always standing out with my curious questions. åsa . . you are a great lunch spinning partner and I cannot see a panda carton without thinking of you. Emil, for the times you stood in for me at course lab. Nisse, for push-up battles in the BMC gym. Huan, it has been great teaching with you. Christian D, for all the cooking inspiration. Magnus, for doing fikas and lunches a little bit funnier. Rickard, for being a much better chair of the PhD association board than I ever was. Tobias, for support and good advice about thesis writing during lunch spinning. My fabulous friends outside work: Saramin and André, our life had been indescribable boring without you. Sara-Lina, if I was a man I would love to be yours :). Pia, thank you for

47

being openhearted and sincere. Karin Schannong, with your honesty, humor and intellect you constantly give me new perspectives of life. Jenny, for en-couraging me to start doing my PhD, even though it was a great privilege working with you at GE. Bettan och Johanna, Three for All-All for One. It’s just the way it has always been and will always be.

My family: Mamma, for your endless love, help and support. You are the best grand-mother the boys ever could imagine.

Pappa, thank you for always believing in me. Chrisse, for all the support, feedback and good advice. You are inspiring. My sister Elin, for being caring, loving, and understanding. Nail, Jacob, Sara, Tanja, Henrik, Ida, Ebba, faster Zorica, Gurkan, Ingegerd. My sweet cousin Nina, her boyfriend Georgos and my aunt Evaggelio for coming to Sweden now and then and taking care of us. You are the best super nannies in this world, σ’αγαπώ. Susi, you know I love you, thank you for keeping me alive in your own crazy, hilarious way during the writing of this thesis. Val-entin and Anita, my super parents in law for all the support over the years. Farmor Carin, when it’s hard I’m thinking of your encouraging words and I get a little bit stronger. I know how much you had wanted to read this thesis because I know how proud you had been. I’m sorry I couldn’t make it in time.

Finally, to whom I dedicate this thesis: Pavel, since the day I met you I have been amazed by your intelligence, con-fidence and incredible discipline. You always give me new insights and per-spectives of Life. Tack för ditt oändliga stöd och för att du vill dela ditt liv med mig.

David and Viktor, you are the most Precious in this world. Jag älskar er.

Angelica, Uppsala 2014

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1115

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science andTechnology, Uppsala University, is usually a summary of anumber of papers. A few copies of the complete dissertationare kept at major Swedish research libraries, while thesummary alone is distributed internationally throughthe series Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology.(Prior to January, 2005, the series was published under thetitle “Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology”.)

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