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New Approaches to combat the HIV infection.
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Dept. of Pharmaceutical chemistryL. M. College of PharmacyNavrangpura, Ahmedabad-09.
Hardik Mistry
1
• Viruses are small infectious agents consisting essentially of nucleic acid (either RNA or DNA) enclosed in a protein coat called capsid.
• The coat plus the nucleic acid core is termed as the nucleocapsid.
• Some viruses have, in addition, a lipoprotein envelope, which may contain antigenic viral glycoproteins, as well as host phospholipids acquired when the virus nucleocapsid buds through the nuclear membrane or plasma membrane of the host cell.
• Certain viruses also contain enzymes that initiate their replication in the host cell.
• The whole infective particle is termed as a virion. In different types of viruses the genome may be double or single stranded.
2
• Viruses are intracellular parasites with no metabolic machinery of their own.
• In order to replicate they have to attach to and enter a living host cell- animal, plant or bacterial and use its metabolic processes.
• The binding sites on the virus are polypeptides on the envelope or capsid.
• The receptors on the host cell, to which the virus attaches, are normal membrane constituents like receptors for cytokines, neurotransmitters or hormones, ion channels, integral membrane glycoproteins, etc.
Virus function and life historyVirus function and life history
3
• Viral DNA enters in the host cell nucleus, transcription of this viral DNA into mRNA by host cell RNA polymerase followed by translation of the mRNA into virus-specific proteins.
• Some of these proteins are enzymes that synthesize more viral DNA as well as proteins of the coat and envelope.
• After assembly of coat proteins around the viral DNA, complete virions are released by budding or after cell lysis.
Condit, R. C.: Principles of virology. In Knipe, D. M.; Howley, P. M. (eds): Fundamental Virology, Lipincott Williams & Wilkins, 4th ed., 2001, pp. 19.
Replication in DNA virusesReplication in DNA viruses
4
• Enzymes in the virion synthesize its mRNA or the viral RNA serves as its own mRNA. • This is translated into various enzymes, including RNA polymerase (which directs the synthesis of more viral RNA) and also into structural proteins of the virion.
• Assembly and release of virions occurs as explained above.
Replication in RNA virusesReplication in RNA viruses
5
• The virion in retroviruses contains a reverse transcriptase (virus RNA- dependent DNA polymerase), which makes a DNA copy of the viral RNA.
• This DNA copy is integrated into the genome of the host cell and it is then termed a provirus.
• The proviral DNA is transcribed into both new genomic RNA and mRNA for translation into viral proteins.
• The completed viruses are released by budding and many can replicate without killing the host cell.
Replication in retrovirusesReplication in retroviruses
6
HIV : ‘Human Immunodeficiency Virus‘ Member of the lentivirinae (lenti, meaning “slow”) subfamily of retroviruses.
Only three retroviruses are known to infect humans. HTLV I (Human T Leukemia Virus I) HTLV II (Human T Leukemia Virus II) HTLV III (Human T Lymphotropic Virus) • HTLV III was confirmed to be the etiological virus for the AIDS
Types of HIV: • HIV-1: 3 groups - M (main) (9 subtypes : A-D,H-F,J,K), - N (new or non-M, non-O), - O (outlier).• HIV-2: 6 distinct phylogenetic lineages – Subtypes/clades: A-F.
Rang, H. P.; Dale, M. M.; Ritter, J. M.; Moore, P. K., Antiviral Drugs., Text Book of Pharmacology, Elsevier Science, 5th ed., 2003, pp. 657
Introduction of HIVIntroduction of HIV
7
HIV-1 subtype B is primarily responsible for the AIDS in North America and Western Europe.
• HIV-2 is less prevalent, less pathogenic & slow progression to AIDS and cause lymphadenopathy.. Found in west Africa & India.
Origin of HIV:
1. HIV-1 from cross-species transmission (zoonosis) of a chimpanzee virus to human.
2. HIV-2 from cross-species transmission of a sooty mangabey virus.
• HIV discovered in 1984 by - Luc Montagneir - Pasteur Institute
• HIV is a retrovirus that infects cells of the human immune system (mainly CD4 positive T cells and macrophages) & destroys or impairs their function which results in the progressive depletion of the immune system, leading to 'immune deficiency‘ and AIDS.
8
p17
p24
Viral envelop
Proteases
Integrase
12. Pantaleo, G.; Graziosi, C.; Fuci, A.S. The immunopathogenesis of human immunodeficiency virus infection, N Engl J Med, 1993, 328,327-335
HIV structure and molecular biologyHIV structure and molecular biology
9
• HIV has following types of cgenes encoding for structural proteins. The gag gene provides the physical infrastructure of the virus; The pol provides the basic enzymes by which retroviruses reproduce; The env gene supplies the proteins essential for viral attachment and entry
into a target cell.
• The accessory proteins tat, rev, nef, vif, vpr, and vpu enhance virus production. tat and rev are essential for viral replication.
• A mutation causes the production of alternate accessory protein, from the fusion of tat, rev, and env in some HIV.
The gp120 and gp41 proteins, both encoded by the env gene, enable the virus to attach to and fuse with target cells to initiate the infectious cycle.
15. Mehannna, A. S. Rational of Design of Anti-HIV Drugs, Abraham, D. J.,Burger’s Text Book of Medicinal Chemistry Drug Discovery, A John Wiley and Sons, Inc., 6th ed, 2003, 5, 461.
HIV genomeHIV genome
10
1. Viral Attachment
2. Viral Penetration/Fusion
3. Uncoating
4. Reverse Transcription
5. Integration
6. Viral Latency and Protein Synthesis
7. Cleavage & Viral assembly
8. Budding
Mehannna, A. S. Rational of Design of Anti-HIV Drugs, Abraham, D. J., Burger’s Text Book of Medicinal Chemistry Drug Discovery, A John Wiley and Sons, Inc., 2003, 6th ed, 5, 461-464.
Important steps of HIV life cycleImportant steps of HIV life cycle
11
Virus binding to the host cell membraneVirus binding to the host cell membrane
12
Viral Penetration/FusionViral Penetration/Fusion
13Theboy,com
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Viral DNA formation by the reverse transcriptaseViral DNA formation by the reverse transcriptase
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Integration of viral DNA into the host genomeIntegration of viral DNA into the host genome
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Integration of viral DNA into the host genomeIntegration of viral DNA into the host genome
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Integration of viral DNA into the host genomeIntegration of viral DNA into the host genome
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Assembly of viral proteins to form the virionAssembly of viral proteins to form the virion
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Viral budding out of the host cellViral budding out of the host cell
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• Mode of viral transmission
I. Sexual (primarily receptive anorectal and vaginal intercourse)
II. Parenteral (sharing of blood-contaminated needles by injection-drug
users, needlesticks, intravenous injection with used needles, receipt of
blood products, or organ transplants)
III.Perinatal/Vertical transmission (mother-to-child transmission-15-30%
risk during pregnancy, labor or breastfeeding: prolonged rupture of
membranes, chorioamnionitis, genital infections during pregnancy, preterm
delivery, vaginal delivery, illicit drug use during pregnancy, high maternal
viral load and Breast-feeding)
IV.Infected body fluids (semen and cervical secretions)
HIV infection and its pathological effectsHIV infection and its pathological effects
21
• It targets T4-lymphocyte, a white blood cell that has a central role specifically the CD-4 helper T-cell.
• HIV starts its replication cycle in the host cell with the help of the enzyme Reverse Transcriptase
• Fall from a normal value of 800-1300 cells/cm2 of blood to below 200 which may give threatening illness.
• The suppressed immune system leaves the patient vulnerable to the so-called opportunistic infections by agents that would not harm a healthy person.
• The most common of such infections is pneumonia caused by Pneumocystis carinii which cause most of the clinical complications
Fauci, A. S. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science, 1988, 239, 617-622.
Cellular picture of the infection
Clinical picture of the infection
Immunosuppressive effect
22
• It is observed in the brain , independent of the immunodeficiency are an abnormal proliferation of the glial cells that surround the neurons and lesions resulting from loss of white matter.
• This can give rise to a neurological symptoms such as dementia and multiple sclerosis.
• With HIV virus there is an increased risk of at least three types of human tumors.
Kaposi's sarcoma a rare tumor of blood vessel tissue in the skin or internal organs. carcinoma including skin cancer which are often seen in the mouth or rectum of infected homosexuals. B-lymphomas (tumors originating in B-lymphocytes).
Neurological effect
Carcinogenic effects
23
1
2
3
4
5
6
7
8
9
10
TARGETS
TARGETS
24
N N
N
SS
O
O
O
O
CH3H3C
Cyclotriazadisulffonamide (CADA)
• Cyclotriazadisulfonamide (CADA) (1) was recently shown to inhibit
HIV (as well as human herpesvirus type 7) infection by down-modulation of the cellular CD4 receptor.
• CADA derivatives not alter the expression of any other cellular receptor (i.e., CXCR4, CCR5).
1. CD4 as the primary cell receptor 1. CD4 as the primary cell receptor
25
• CADA is assumed to down-regulate CD4 expression at the post translational level.
• CADA ,it proved synergistic in its anti-HIV activity when combined with NRTIs (i.e., zidovudine, lamivudine, zalcitabine, abacavir), NtRTIs (i.e., tenofovir), NNRTIs (i.e., nevirapine, delavirdine), PIs (protease inhibitors lopinavir, saquinavir, indinavir, nelfinavir,
amprenavir. and ritonavir, The gp41 fusion inhibitor T-20 (enfuvirtide), The CXCR4 antagonist AMD3100, and the Mannose-specific plant lectins from Galanthus nivalis (GNA) and
Hippeastrum hybrid (HHA).4
Mechanism of action
26
• Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) ,the plant lectins derived from GNA (Snowdrop) and HHA (Amaryllis) were shown to interrupt the viral entry process by interfering with the viral envelope glycoprotein gp120.
• When HIV-1 was made resistant to GNA and HHA, several amino acid changes were noted in gp120 but not gp41; occurred at the N-glycosylation sites (at the S or T residues).
• They are used as topical microbicides for the prevention of the sexual transmission of HIV infection.
2. Virus Attachment Inhibitors2. Virus Attachment Inhibitors
27
• Cyanovirin
A potential microbicide to prevent the transmission of HIV and AIDS , isolated from the cyanobacterium Nostoc ellipsosporum.
• Cyanovirin-N has a uniquely high affinity for gp120: it impairs both CD4-dependent and -independent binding of gp120 to the target
cells, it blocks CD4-induced binding of gp120 with CXCR4, and it dissociates bound
gp120 from target cells.
• The aglycons of the glycopeptide antibiotics vancomycin, teicoplanin, and eremomycin, were found to display activity against HIV-1, HIV-2, and Moloney murine sarcoma virus at the lower concentration range.
28
• The Teicoplanin aglycon , interferes with viral entry, probably at the virus adsorption step, because their anti-HIV activity was lost if added at 1-2 h after infection.
• Glycopeptide antibiotic aglycon derivatives could be envisaged as potential lead compounds for application as microbicides against sexual HIV transmission.
29
• BMS- 378806 : A new class of HIV-1 attachment inhibitors [prototype: 4-benzoyl-1-[4-methoxy-1H-pyrrolo[2,3-b]- pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS- 378806) (4)] that interferes with CD4-gp120 interactions.
• It binds directly to gp120 at a stoichiometry of approximately 1:1, with a binding affinity similar to that of soluble CD4.
NNH
O
N
N
O
OCH3
CH3
O
BMS-378806 4
30
• The potential target site of BMS- 378806 was localized to a specific region within the CD4 binding pocket of gp120.
• The 4-resistant variants have been isolated with amino acid substitutions (M426L and M475I) in the CD4-binding pocket of gp120, confirming that BMS- 378806 targets gp120.
• BMS- 378806 displays many favorable pharmacological traits such as Low protein binding, Minimal human serum effect on anti- HIV-1 potency, Good oral bioavailability in animal species, and A clean safety profile in initial animal toxicology studies.
31
• BMS-488043 : This Virus attachment inhibitors may be effective in vivo in HIV-1-infected patients (5), which is structurally closely related to BMS- 378806 .
• BMS- 488043 is assumed to bind to the HIV-1 glycoprotein gp120 and to induce or block conformational changes in the gp120.
• The compound was well tolerated, and no serious adverse events were noted.
NNH
O
N
N
O
OCH3
O
BMS-488043 5OCH3
32
• To enter the cells following binding with the CD4 receptor, the HIV
particles must interact, again through the viral envelope glycoprotein gp120, with the CXCR4 or CCR5 coreceptor.
• CXCR4 is the coreceptor for Tlymphotropic (or X4) HIV strains, whereas CCR5 is the coreceptor for macrophage (M)-tropic (or X5) HIV strains.
• CXCR4 normally functions as the receptor for the chemokine SDF-1 (stromal cell derived factor), and CCR5 does so for RANTES (regulated upon activation, normal T-cell expressed, and secreted) and MIP-1R and -1‚ (macrophage inflammatory proteins), and accordingly, these chemokines inhibit the infectivity of X4 and X5 HIV strains, respectively.
3. CXCR4 and CCR5 Antagonists 3. CXCR4 and CCR5 Antagonists
33
• Bicyclam AMD3100 (6) is low-molecular-weight CXCR4 and CCR5 antagonists
• Compound 6 is not truly specific for CXCR4. It does interact with any other CXCR or CCR receptor and blocks X4 HIV-1 replication through CXCR4 antagonization.
N N HNNH
HNNH NH HN
AMD3100 6
34
• During phase I clinical studies AMD3100 caused a significant enhancement of the white blood cells (WBC) counts in human volunteers.
• It mobilize hematopoietic stem cells from the bone marrow into the bloodstream, and because this effect proved synergistic with the action of G-CSF (granulocyte-colony stimulating factor), so AMD3100 is now being pursued (in phase II clinical studies) for stem cell mobilization and transplantation in patients with multiple myeloma or non-Hodgkin lymphoma.
• CXCR4 antagonists such as AMD3100 are able to suppress in vivo replication of X4 or dualtropic X4/R5 HIV strains.
35
• Structure-activity relationship (SAR) studies revealed that the bis-macrocyclic structure is not an absolute prerequisite for anti-HIV activity.
• For example: AMD3465 (7), an AMD3100 derivative ,proved to be as potent, as AMD3100 as both a CXCR4 antagonist and inhibitor of X4 HIV strains.
• Also, KRH-1636 (8), another CXCR4 antagonist, with anti-HIV activity similar to that of AMD3100.
• KRH-1636 appears to be duodenally absorbable, at least in rats, which means that it may be orally bioavailable.
N NH
NH
HNNH
AMD3465 7
N
NH
O
HN
O
NH
CH3
NH
H2N NH
N
KRH-1636 8
36
• TAK-779 (9): The CCR5 antagonists, the quaternary ammonium derivative was the first nonpeptidic molecule which block the replication of the M-tropic R5 HIV-1 strains (in the nanomolar concentration range) by interaction with CCR5.
• The binding site for TAK-779 has been identified within the transmembrane (TM) helices 1, 2, 3, and 7 of CCR5.23
• TAK-779 is not orally bioavailable and provokes irritation at the injection site.
H3CO
HN
N
CH3
CH3
O
Cl-
TAK-779 9
37
• SCH 351125 (SCH-C) (11) was the first CCR5 antagonist to be advanced to clinical studies.
• SCH 351125 has potent activity in vitro against primary HIV-1 isolates using CCR5 as their cell entry coreceptor.
• SCH-351125 bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by the transmembrane (TM) helices 1, 2, 3, and 7.
N
N
N O
N
Br
O
OSCH-C(SCH 351125) 11 38
• Spirodiketopiperazine E913 (12).
• E913 specifically blocks the binding of MIP-1R to CCR5, the MIP-1R-elicited Ca2+ flux, and the replication of both laboratory and primary R5 HIV-1 strains, as well as various multidrug-resistant monocyte/ M-tropic R5 HIV-1 strains.
• From the spirodiketopiperazine class of compounds, E913 was recently quoted as binding only partially to CCR5 while exhibiting much greater anti-HIV activity.
O
O
N
NH
N
O
O
39
• UK-427,857 (13): CCR5 antagonist, has recently been selected as a clinical development candidate drug for the treatment of HIV infection.
• UK-427,857 was reported to display excellent potency against a isolates utilizing CCR5 for cell entry (at IC90 < 10 nM) while being inactive against CXCR4-tropic viral isolates.
F
F
O
HN
N
H
H
N N
N
UK-427857 13
40
• 1,3,4-trisubstituted pyrrolidine : CCR5 receptor antagonists have possess oral bioavailability and/or potent anti-HIV activity.
• A representative congener of this 1,3,4- trisubstituted pyrrolidine series is 14. The compound’s site of interaction with CCR5 has been mapped to a cavity, near the extracellular surface, formed by the TM helices 2, 3, 6, and 7.
N
N
O
2
1
34
5
1,3,4-Trisubstituted pyrrolidine 14
41
• CMPD 167, CCR5 antagonists are efficacious in vivo against CCR5-using virus strains, designated previously as MRK-1 (15).
• This compound caused a rapid and substantial (4- to 200-fold) decrease in plasma viremia in rhesus macaques chronically infected with simian immunodeficiency virus (SIV).
• It is used as a topical microbicide to prevent HIV-1 sexual transmission because viral replication could be partially inhibited by it.
H3C
CH3H3C
O
CH3
N
NN
CH3
F
MRK-1CMPD 167
15
42
• The interaction of the X4 or R5 HIV-1 envelope gp120 with the
coreceptors CXCR4 or CCR5, respectively, is followed by a spring-loaded action of gp41 that then anchors through its amino terminus into the target cell membrane and, in doing so, initiates the fusion of the viral envelope with the cellular plasma membrane.
• During the process, hydrophobic grooves on the surface of the coiled coil gp41 ectodomain become available for binding with extraneous inhibitors,
that are virus cell fusion inhibitors.
4. Virus-Cell Fusion Inhibitors 4. Virus-Cell Fusion Inhibitors
43
• For example: Enfuvirtide (T-20, DP-178, pentafuside, Fuzeon) (16), a synthetic, 36-amino-acid peptide corresponding to residues 127-162 of gp41 or residues 643-678 of the gp160 precursor.
44
• Drawbacks of Enffuvirtide
Enfuvirtide has to be administered twice daily by subcutaneous injection. This inevitably leads to injection- site reactions including erythema, induration, and nodules, and cysts.
Another problem is the production cost for a 5000 Da molecular mass peptide such as enfuvirtide.51
Even enfuvirtide-insensitive HIV-1 variants may exist in an enfuvirtide-naive population, and this may allow virus escape from drug.
45
• Tris-functionalized 3,2’,2’’-terphenyl derivatives (17) could serve as effective mimics of the exposed N-helical regions of the transient gp41 intermediate and thus potentially trap this structure prior to the six-helix bundle formation, which is required for virus-cell fusion.
• Compound 17 was found to inhibit HIV-1 mediated cell-to cell fusion but only at a relatively high concentration (IC50 15 μg/mL).
O
OH
OCH3
CH3
CH3H3C
CH3
H3C
HO
O
Terphenyl derivative 17
46
• Zidovudine (AZT, Retrovir),
• Didanosine (ddI, Videx),
• Zalcitabine (ddC, Hivid),
• Stavudine (d4T, Zerit),
• Lamivudine (3TC, Epivir) (also marketed in combination with zidovudine under the trademark Combivir and with abacavir under the trademark Epzicom), and
• Abacavir (ABC, Ziagen) (also marketed in combination with zidovudine and lamivudine under the trademark Trizivir),
• Emtricitabine (2’,3’-dideoxy-3’-thia-5-fluorocytidine, (-)-FTC, previously referred to as Coviracil and now marketed as Emtriva) is the seventh 2’,3’- dideoxynucleoside analogue that has been officially approved (for the treatment of HIV infections.
5. Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
5. Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
47
N
N
NH2
O
O
HO
Zalcitabine
N
N
NH2
O
O
S
HO
Lamivudine
HN
N
O
O
O
HO
CH3
Stavudine
HN
N
O
O
O
HO
CH3
N3
Zidovudine
HN
N
N
O
O
HO
Didanosine
48
• Emtricitabine can be conveniently administered as a once-daily dose of 200 mg for long term clinical use in HIV-1-infected individuals.
• In studies through 60 weeks, once daily emtricitabine, combined with once-daily didanosine and efavirenz, demonstrated durable and superior virologic efficacy and tolerability, compared to twice-daily stavudine and once-daily didanosine and efavirenz.
S
O
HO
N
N
NH2
F
O
Emtricitabine [(-)FTC] 18 49
• Emtricitabine has been considered an “ideal drug candidate” because
It shows synergism with other antiretrovirals,
Excellent tolerability,
A long intracellular half-life (supporting the once-daily dosing), and,
In comparison with lamivudine, 4- to 10-fold higher in vitro potency against HIV.
50
• Amdoxovir ((-)-‚-D-2,6- diaminopurine dioxolane, DAPD, 21):
New purine -based 2’,3’-dideoxynucleoside analogues
• DAPD is converted by adenosine deaminase to dioxolane guanine (DXG) which is then phosphorylated intracellularly to DXG 5’-triphosphate, the active metabolite.
• DXG 5’-triphosphate (DXG-TP) acts as an alternative substrate/ inhibitor of the HIV-1 reverse transcriptase.
• DAPD/DXG has proven active against HIV-1 mutants resistant to zidovudine (M41L/D67N/K70R/T215Y/K219Q) and lamivudine (M184V)73 but has decreased activity against viruses carrying the K65R and Q151M mutations.
N
NN
N
NH2
O
O
HO
Amdoxovir (DAPD) 21
NH2
51
• The nucleotide reverse transcriptase inhibitors (NtRTIs) such as Adefovir [9-(2-phosphonylmethoxyethyl) adenine (PMEA)] and Tenofovir [(R)-9-(2- phosphonylmethoxypropyl)adenine (PMPA)]
are already equipped with a phosphonate group and, therefore, only need two phosphorylation steps to be converted to the active metabolites (PMEApp and PMPApp, respectively).
• The metabolites then serve as alternative substrates in the reverse transcriptase reaction, where upon their incorporation they act as chain terminators.
6. Nucleotide Reverse Transcriptase Inhibitors (NtRTIs)
6. Nucleotide Reverse Transcriptase Inhibitors (NtRTIs)
52
• The NtRTIs Adefovir and Tenofovir are active not only against HIV but also against hepatitis B virus (HBV), because HBV uses for its replication a reverse transcriptase that is quite similar to that of HIV.
• They have been officially approved, in their oral prodrug forms Adefovir dipivoxil [bis( pivaloyloxymethyl )-PMEA, Hepsera] (22) and Tenofovir disoproxil [bis( isopropyloxycarbonyloxymethyl )- PMPA] fumarate (Viread) (23), for the treatment of HBV and HIV infections, respectively.
N
NN
N
OP
OO
O
NH2
OH2C
OH2C
C
C
O
O
(H3C)3C
(H3C)3C
Adefovir dipivoxil 22
N
N N
N
OP
OO
O
NH2
OH2C
OH2C
C
C
O
O
O
O
Tenofovir disoproxil fumarate 23
H3C
(H3C)3C
(H3C)3C
HOOC
COOH
53
• Both the d4T (Stavudine) and TDF (Tenofovir) arms showed a similar, high virological response. However, lipid abnormalities (increase in triglyceride and cholesterol levels) were significantly lower in the TDF arm than in the d4T arm.
• Also, the toxicities (peripheral neuropathy, lipodystrophy, lactic acidosis, pancreatitis) associated with mitochondrial dysfunction through week 96 were markedly lower in the TDF arm than in the d4T arm, while both arms showed a similar renal safety profile.
• Although in an animal model for HIV transmission through breast-feeding topical administration of lowdose TDF did not protect infant macaques against multiple oral exposures of simian immunodeficiency virus (SIV),
54
• Another indication for the clinical use of tenofovir disoproxil fumarate is (lamivudine-resistant) chronic hepatitis B in HIV/HBV- coinfected patients.
• Several studies have demonstrated that TDF is very effective in reducing HBV DNA levels in HIV/HBV- coinfected patients carrying either wild-type or 3TC-resistant [YMDD variant (rt M204I/V)] HBV.
• Noteworthy is that TDF treatment for 12 months in patients coinfected with HIV and 3TC-resistant HBV was not associated with the emergence of HIV- or HBV-specific resistance mutations.
55
• More than 30 structurally different classes of compounds have been
identified as NNRTIs, that are targeted at a specific, allosteric (i.e., nonsubstrate binding) site of the reverse transcriptase.
• The “first-generation” NNRTIs are notorious for rapidly eliciting virus drug resistance, especially when used singly.
• The most common mutations occurring in the clinical setting following the use of NNRTIs are K103N and Y181C.
• Therefore, attempts have been made to develop “second-generation” NNRTIs that are resilient to such drug resistance mutations.
• “first-generation” NNRTIs are Nevirapine and Delavirdine while “Second-generation” NNRTIs are Efavirenz .
7. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
7. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
56
• DAPY derivatives
1. TMC 125 (Etravirine) (27) is an investigational NNRTI with potent activity against HIV-1 strains resistant to the currently available NNRTIs.
• It is under the advance clinical development.
N
O
N
N NH
N
BrNH2
Etravirine (Tmc125) 27
57
2. Rilpivirine
more active than NVP, EFV and dapivirine against wild-type HIV-1 and all single and double mutants tested .
• E-form was superior in terms of antiviral activity with respect to the corresponding Z-isomer.
• Rilpivirine was not mutagenic,no relevant side effects, had no effect on cardiovascular, pulmonary, electrophysiological and behavioral parameters in dog.
• It is also under the advance clinical development.
HN
N
N NH
N
Rilpivirine
CN
58
• Dihydropyrazinones
• The cyano substituent in the 4-position of the aniline moiety was the optimal choice,
• The presence of a 6-methyl group in the dihydropyrazinone ring cause a favorable effect on activity against mutants.
• Introduction of methyl substituents on the benzene ring and at position- 6 of the dihydropyrazinone ring on the left wing increased the activity against both HIV wild-type and drug-resistant mutants.
• Compounds with X = SO2 were slightly superior to those with X = S, which was only confirmed for the Y181C mutation.
N
N
NH
X
R
R1
OR2
R335
21
6
4
59
• Benzophenones• These compounds proved to be much more potent than NVP and
DLV, and equivalent to EFV against both wild-type HIV-1 and the Y181C mutant strain.
R1
R2
O
Cl
O
O
HN
SO2NH
23 GW4511 R1=CF3, R2 =F24 GW4751 R1=H, R2=CN
F3C
F
O
Cl
O
O
HN
25 GW3011
O SO2NH
60
• 2-Quinolones
• The quinolones were demonstrated to be potent inhibitors of wild-type (HIV-1 IIIB) and NVP-resistant strains in MT-4 cell assays.
• R = F; Cl as substituents were optimal for antiviral activity in MT-4 cells.
• Replacement of R with CH3 and OCH3, led to much less active compounds.
• Introduction of a cyclopropylethynyl group as the R2 substituent led to the more potent compounds
NH
X
Cl R
O
61
• Indolyl aryl sulfone
• The iodole derivatives bearing the 3-[(3,5-dimethylphenyl) sulfonyl] moiety (52) displayed high activity and selectivity not only against the HIV-1 wild-type strain, but also against the Y181C and K103N-Y181C viral variants and the EFV-resistant (EFVR) mutant K103R-V179D-P225H.
HN
Cl
O
NH2
SO2
R1
R2R3
62
• Capravirine analogues
• Capravirine retained activity against HIV-1 strains carrying the K103N mutation in their reverse transcriptase.
• Replacement of ethyl in position-4 of the imidazole nucleus with an iso-propyl group led to more potent derivatives.
• The activity of the compounds was also affected by the group at the 5-position of the imidazole ring in the order 3,5-(CH3)2-phenyl > cyclohexyl > phenyl group
• The alkoxymethyl substituent at the 1-position of imidazole ring showed higher activity than compounds containing a 4-pyridylmethyl group at the same position.
S
N
N
O
O
H2N
CH3
H3C
Cl
Cl
N
Capravirine (S-1153,AG1549) 25
1
2
34
5
63
• Pyridones• in vitro activity profile of these new molecules, and in particular of
compound 118, was better than that of EFV.
HN
X
O
N
111 X= H2112 X = O
HN
X
O
N
117 X= O118 X = H2
CN
64
• Integration of the proviral DNA into host cell chromosomal DNA is an
essential step in the viral replication cycle.
• This process is mediated by the viral integrase, and because there is no cellular homologue for this enzyme, it has been considered an attractive target for HIV therapeutics.
• Numerous small-molecule HIV-1 integrase inhibitors have been described, the two most predominant classes of inhibitors being the catechol containing hydroxylated aromatics and the diketo acid containing aromatics.
8. HIV Integrase Inhibitors8. HIV Integrase Inhibitors
65
• Catechol containing hydroxylated aromatics
• An example is L- chicoric acid (28).
• Integrase was identified as the molecular target for the action of L- chicoric acid, since a single amino acid substitution (G140S) in the integrase rendered the corresponding HIV-1 mutant resistant to L- chicoric acid.
• It was later shown that the G140S mutation confers resistance not only to L- chicoric acid but also to the diketo acid L-731,988 and furthermore attenuates the catalytic activity of the enzyme.
HO
HO
O
O
OOH
O
O
O
OH
OH
L-Chicoric acid 28
HO
66
• In our hands, however, HIV-1 integrase carrying the G140S mutation appeared to be as sensitive to the inhibitory effect of L- chicoric acid as the wild-type integrase.
• Upon repeated passages of the virus in the presence of L- chicoric acid, mutations were found in the viral envelope glycoprotein gp120 but not in the integrase.
• HIV strains resistant to polyanionic compounds showed cross-resistance to L- chicoric acid, and time-of addition experiments further confirmed that the primary site of interaction for L- chicoric acid is the virus adsorption stage rather than the integrase.
67
• The structure of the HIV-1 integrase core domain complexed with the inhibitor 5CITEP [1-(5-chloroindol- 3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl) propenone] has been described as a platform for the structure-based design of novel HIV-1 integrase inhibitors.
C
ON
HN
N
N
HO NH
Cl
5CITEP
• Diketo acid containing aromatics.
68
F
NCOOH
O O
L-731,988
O
COOH
O
O O
L-708,906
O
O
OH
O
OH
O
O
HO
Granulatine
HN
HN
O O
OH HO
Salicylhydrazine
COOHCHO
OO
Integric acid
69
• There are number of diketo acids such a L-708,906 (29) and L-731,988 (30) as inhibitors of the integrase-mediated strand transfer reaction (which is responsible for the covalent linkage of the viral DNA 3’ ends to the cellular DNA).
• The diketo acid derivative 29 and 30 were also found to inhibit HIV-1 replication in cell culture .
• The proviral DNA integration is indeed the target of action for the diketo acids in cell culture.
N
F
O O
O
OH
L-731,988 30
O
O O
O
OHO
L-708,906 29 70
• S-1360 (31) actually represents the first integrase inhibitor to reach clinical studies.
• S-1360 would inhibit the HIV-1 integrase at an IC50 of 20 nM and the HIV-1 replication at an EC50 of 140 nM while its CC50 would be 110 μM, thus achieving a therapeutic index of almost 1000, the highest selectivity.
• The mechanism of action of the diketo acids may be based on an interaction between the carboxylate group of the diketo acids or the isosteric heterocycle in the two other compounds and metal ion(s) in the active site of the integrase, resulting in a functional sequestration of these critical metal cofactors.
O
O OH
F
N
NHN
S-1360 31
71
• Diketo acids could be classified into two groups:
Those similarly potent in the presence of magnesium and Those potent in manganese and relatively ineffective in the presence of
magnesium.
• Both the aromatic and the carboxylic or tetrazole functions of the diketo acids determine their metal-chelating selectivity.
• Starting from 30, several additional 4-aryl-2,4-dioxobutanoic acid derivatives have been described as HIV-1 integrase inhibitors.
• Also, new azido-containing aryl diketo acids have been described as HIV-1 integrase inhibitors capable of conferring antiviral protection in HIV-infected cells.
72
• 8-hydroxy[1,6]-naphthyridines.
Cause Inhibition of the strand transfer reaction of the integration process.
• The naphthyridine shown (32) inhibits strand transfer catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 μM.
• While no cytotoxicity is exhibited in cell culture at e12.5 μ M, and a good pharmacokinetic profile was displayed when dosed orally to rats.
N
O
N
N
OH
S
O
O
8-Hydroxy-[1,6]naphthyridine 32 73
• Recently, an entirely new class of HIV integrase inhibitors was 5H-pyrano- [2,3-d:-6,5-d¢]dipyrimidines (PDPs).
• The most potent congener of this series, 5-(4-nitrophenyl)-2,8-dithiol-4,6- dihydroxy-5H-pyrano[2,3-d:-6,5-d’]dipyrimidine (V-165) (33), inhibited the replication of HIV-1 at an EC50 of 8.9 μM, which is 14-fold below the cytotoxicity threshold.
N
N O N
N
OHOH
HSSH
NO2
Pyranodipyrimidine (PDP) (V-165) 33
74
• At the transcriptional level, HIV gene expression may be inhibited by compounds that interact with cellular factors (such as NF-B) that bind to the LTR promotor and that are needed for basal-level transcription.
• These compounds specifically inhibit the transactivation of the HIV LTR promotor by the viral Tat (trans-activating) protein.
• These compounds inhibit HIV-1 replication in both acutely and chronically infected cells, through interference with the transcription process that attributed to inhibition of Tat (or other transactivators).
9. Transcription (Transactivation) Inhibitors9. Transcription (Transactivation) Inhibitors
75
K37 [7-(3,4-dehydro-4-phenyl-1-piperidinyl)- 1,4-dihydro-6-fluoro-1-methyl-8-trifluoromethyl- 4-oxoquinoline-3-carboxylic acid;
Temacrazine [1,4-bis(3-(6-oxo-6H-v-triazolo[4,5,1-de]acridin-5-ylaminopropyl) piperazine];
Flavopiridol, a cyclin-dependent kinase (CdK) inhibitor;
EM2487, a natural product from Streptomyces; and
CGP 64222, a 9-mer peptoid structurally reminiscent of the amino acid 48- 56 sequence RKKRRQRRR
of Tat. peptoid CGP 64222 owes its anti-HIV activity in cell culture primarily to an interaction with CXCR4, the coreceptor for X4 HIV strains.
76
• A 6-aminoquinolone, WM5 (34), was recently shown to inhibit HIV-1 replication in acutely infected as well as chronically infected cells.
• This aminoquinoline was found to efficiently bind to TAR RNA and to inhibit Tat mediated LTR-driven transcription.
N
N
N N
H2N
CH3
O O
OH
Aminoquinolone WM5 34
77
• Recently prooved that the co transcriptional capping of HIV mRNA is stimulated by Tat and consequently its binding to the capping enzyme.
• These findings implicate capping as an elongation checkpoint critical to HIV gene expression and thus corroborate that S-adenosylmethionine- dependent methylations play an important role in the Tat dependent transactivation of transcription from LTR.
• They also explain the inhibitory effects of S-adenosylhomocysteine hydrolase inhibitors, such as neplanocin A and 3-deazaneplanocin A (35), on Tat dependent transactivation and HIV replication.
N
XN
N
NH2
HO
OH OH
X= N2 Neplanocin AX= CH: 3-Deazaneplanocin A 35
78
• After transcription the unspliced (or partially spliced) HIV mRNA has to be transported from the nucleus into the cytoplasm in order to be translated to viral proteins.
• This export is promoted by the HIV-1 Rev (regulator of expression of viral proteins).
• Nuclear export of Rev is mediated by its leucine-rich nuclear export signal (NES). NES uses the export factor CRM1 to export viral mRNA from the nucleus to the cytoplasm.This process can be blocked by a small molecular- weight molecule, PKF 050-638 (36), that specifically inhibits CRM1-NES complex formation and, hence, Rev-mediated nuclear export.
N
N
N
Cl
NH2
O
CH3
O
PKF 050-638 36
79
• N-aminoimidazole derivatives (NAIMS), structurally analogous to Capravirine (S-1153, AG1549) have been found to inhibit HIV replication through one of the following types of interaction.
(i) the classical NNRTI type of action. (ii) an as yet unidentified target of action; and (iii) combination of type i and type ii actions.
N
N
HN
R
Y
X2
X1
N-aminoimidazoles (NAIMS) 37
X1,X2 ; Halogen ,AlkylR :Alkyl , ArylY: SH , H
80
• Pyridine oxide derivatives (prototypes JPL-32, JPL-88, and JPL-133) (38) like the NAIMS, can exhibit same behavior.
• Some of the pyridine oxides behave as typical NNRTIs.
S
R1
R2
N Y1
Y2
Y3
Y4
X3
X2X4
X5 X1
+
O -
Pyridine oxides JPL-32,-88 and-133
JPL-32 JPL-88 JPL-133
X1 Cl CH3 CH3
X2 Cl H H
X3 Cl CH3 H
X4 Cl H CH3
X5 Cl CH3 H
Z H H H
R1 O - -
R2 O O -
Y1 H H H
Y2 H H H
Y3 H H H
Y4 H H Cl
81
• The most active congener from this series, JPL-133, has an EC50 of 0.05 μg/mL against HIV-1 and a selectivity index of approximately 760 in cell culture.
• For other pyridine oxides such as JPL-32 and JPL-88, experiments revealed a postintegration step in the HIV replicative cycle as the most likely target of action.
• The mode of action of JPL-32 appeared to be reminiscent of that of K-37 because JPL-32, like K-37, was active in both acutely and chronically HIV-1-infected cells.
• JPL- 32 also inhibited the Tat-mediated HIV-1 mRNA transcription from HIV-1 LTR, thus clearly indicating that its target of action is located at the transcription transactivation level.
82
• The HIV protease is responsible for the cleavage of the gag and gag-pol precursor polyproteins to the structural proteins (p17, p24, p7, p6, p2, p1) and functional proteins [protease (p11), reverse transcriptase (p66/p51), and integrase (p32)], thereby securing maturation and infectivity of the progeny virions.
• HIV protease inhibitors will interfere with this late stage of the viral replication cycle and prevent formation of infectious virus particles.
10. HIV Protease Inhibitors10. HIV Protease Inhibitors
83
• All protease inhibitors (PIs) that have been licensed for the treatment of HIV infections, namely,
Saquinavir (Fortovase, Invirase), Ritonavir (Norvir), Indinavir (Crixivan), Nelfinavir (Viracept), Amprenavir (Agenerase) (and its phosphate derivative, Lexiva), Lopinavir (Kaletra, lopinavir with ritonavir at a 4:1 ratio), and Atazanavir (Reyataz),
N
N
NH
HN O
O
HN
NH
O
O
O
O OH
Atazanavir (BMS-232632) 39
84
• The Aza-dipeptide analogue Atazanavir (39), the latest and seventh PI
To be approved for clinical use, Combines a favorable resistance profile distinct from that of the other PIs, A favorable pharmacokinetic profile allowing once-daily dosing.
• Nelfinavir-, Saquinavir-, and Amprenavir-resistant HIV-1 strains remained sensitive to Atazanavir, while Indinavir- and Ritonavir-resistant viruses showed 6- to 9-fold changes in sensitivity to atazanavir.
• Conversely, Atazanavir-resistant (N88S, I84V) virus, selected upon repeated passage of the virus in the presence of the compound, remained sensitive to Saquinavir but showed various levels of cross-resistance to Nelfinavir, Nndinavir, Ritonavir, and Amprenavir.
85
• TMC 126 (UIC-94003) ,Compound 40 is a peptidomimetic PI containing a bis-tetrahydrofuranyl urethane and 4-methoxybenzenesulfonamide (and thus structurally related to amprenavir).
• It was reported to be extremely potent against a wide spectrum of HIV-1 strains (EC50 ) 0.3-0.5 nM), whether resistant to other PIs or not.
NH
N
S
OH CH3
CH3
O
O
OCH3
O
O
O
O
UIC-94003 (TMC-126) 40 86
• Upon passage of HIV-1 in the presence of 40, mutants carrying a novel active-site mutation, A28S, emerged.
• Modeling studies revealed close contact of 40 with the main chain of the protease active-site amino acid residues D29 and D30 different from that of the other PIs, and this may be important for its potency, particularly against drug-resistant HIV-1 variants.
• TMC 114, like TMC 126, has an excellent activity profile against HIV variants that are highly resistant to current PIs.
87
• Tipranavir (PNU-140690) (41) is a nonpeptidomimetic inhibitor of the HIV protease,which, shows little cross-resistance to the peptidomimetic PIs.
• Tipranavir retained marked activity against HIV-1 isolates derived from patients with multidrug resistance to other Pis.
• Tipranavir drug levels can be markedly increased (“boosted”) by coadministration with ritonavir.
O O
OH
NH
S
O O
N F
F
F
Tipranavir (PNU-140694) 41
88
• During the reverse transcription process that converts the single-stranded
viral RNA into double-stranded (pro)viral DNA, a RNA‚DNA heteroduplex (hybrid) is formed.
• The RNA strand of this heteroduplex must be cleaved by the ribonuclease H (RNase H) component before the remaining (-)- DNA strand can be duplicated to give the doublestranded (pro)viral DNA that is then integrated into the host cell genome.
• RNase H has been considered as an attractive target for the development of new antiretroviral agents.
HIV Ribonuclease H Inhibitors HIV Ribonuclease H Inhibitors
89
• It is feasible to specifically inhibit HIV-1 Rnase H by, for example, novel Diketo acids, such as 4-[5- (benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid (42), and N-hydroxyimides such as 2-hydroxy-4H-isoquinolone- 1,3-dione (43).
• In both cases, binding of the compound was (much) stronger in the presence of Mn2+ compared to Mg2+.
N
O
OH
O2-hydroxyisoquinoline-1,3(2H,4H)-dione
O
HN S
O O
O
OH
4-(5-benzamidothiophen-2-yl)-2,4-dioxobutanoic acid42
90
Inhibitors of viral entry
• It is most ideal approach to inhibit HIV binding to the T-cell
• It can neutralize the virus in circulation.
• The initial work on vaccine development focused on iso-typic variants of the HIV envelope glycoprotein gp120 obtained by recombinant DNA techniques due to concern about the safety of live/attenuated vaccines.
• The gp120 glycoprotein is a coat protein, and if great care is taken, a virus-free vaccine is obtainable.
• Moreover, glycoprotein gp120 is a primary target for neutralizing antibody associated with the first step in HIV infection.
HIV vaccinesHIV vaccines
91
• Today it is facilitated the design of vaccination regimens that elicit specific immune responses and effector mechanisms.
• The importance of CD8+ CTL responses in controlling HIV and SIV viremia has led to a series of vaccine candidates that effectively induce these responses.
• gp120-derived vaccines induced little cell-mediated immunity and strong antibody response in T-cell lines, but failed to neutralize virus derived from peripheral blood mononuclear cells.
• Recombinant attenuated vaccinia virus expresses key viral envelope protein followed by a booster of soluble envelope protein derived from HIV.
• It produced a good humoral and CMI response and IgA antibodies in animal models.
92
• AIDSVAX25• AIDSVAX from VaxGen Inc. is a preventive vaccine made up of synthetic gp120.
Two Phase III clinical trials were initiated. Failed due to lack of adequate protection.
• DNA VACCINE26• In recent years a new type of vaccine, created from an infectious agent's DNA
called DNA vaccination.• It works by insertion (and expression, triggering immune system recognition) into
human or animal cells, of viral or bacterial DNA.• As of 2006, DNA vaccination is still experimental, but shows some promising
results.• However, the nature of the disease people infected with HIV develop only low
titers of neutralizing antibody and that presents problem for vaccine development.
93
