Mycobacterium tuberculosis Type-II NADH dehydrogenase (NDH-2) Catalytic Mechanism and Mode of Action of NDH-2 Inhibitors

Takahiro Yano, Miriam Rahimian, Kawalpreet K. Aneja, Norman Schechter, Charles P. Scott, and Harvey Rubin University of Pennsylvania, Philadelphia USA (Supported by NIH and TB Alliance)

1. Abstract 3. Catalytic mechanism of NDH-2

2. OxPhos of Mtb

4. Mode of action of NDH-2 inhibitors

5. Conclusion

Type-II NADH-quinone (Q) oxidoreductase (NDH-2) catalyzes electron transfer from NADH to the Q-pool and plays an essential role in the oxidative phosphorylation system (OxPhos) of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial OxPhos makes this enzyme an attractive target for antibiotic development. In this study, the catalytic mechanism of Mtb NDH-2 was investigated in detail by comparing the kinetics of the NADH-Q reductase reaction to those of the NADH-thio-NAD+ transhydrogenase reaction catalyzed by NDH-2, which follows a one-site ping-pong mechanism. We obtained evidence that the Mtb NDH-2 catalyzes electron transfer from NADH to Q by a non-classical two-site ping-pong mechanism where the substrate Q binds to a site distinct from the NADH-binding site. Furthermore, a study of effects of quinols on Mtb NDH-2 catalytic activity strongly suggested the presence of two Q-binding sites, one binds to oxidized Q and the other preferably binds to reduced Q.

Based on the catalytic mechanism determined in this study, the mode of action of NDH-2 specific inhibitors that had been identified through a high throughput screening was investigated. We indentified two classes of inhibitors that interact with the Q-binding sites by different inhibition modes. One class of inhibitors compete with substrate Q and the other class shows a uncompetitive inhibition pattern with Q. The results suggest that the two-quinone sites in NDH-2 may constitute a unique target for the development of selective antibiotics.

Mtb is an obligatory aerobe and acquires an energy (ATP) through the oxidative phosphorylation system (OxPhos, Figure 1)1. The NDH-2 is a membrane-bound, single polypeptide enzyme containing a FAD as a cofactor and plays an essential role in oxidizing NADH with menaquinone to menaquinol. Menaquinol carries electrons/protons within the membrane to a supercomplex composed of cytochrome bc1 reductase and aa3-type cytochrome c oxidase. Electron transport and the reduction of O2 to water are coupled to proton translocation across the membrane establishing an electrochemical proton gradient (∆µH

+) that drives ATP synthase. A second terminal oxidase, cytochrome bd oxidase, functions under low oxygen conditions and can directly oxidize menaquinol with O2. Inhibition of NDH-2 (phenothiazines)2-4, or ATP synthase (Bedaquiline)5 kills the organism, validating the OxPhos as an attractive target of new antibiotics. We have also shown previously that clofazimine (CFZ) interacts with and is reduced by Mtb NDH-26. The reduced CFZ spontaneously reacts with molecular oxygen, producing an superoxide anion (O2

-) and is re-oxidized (Figure 3). This NDH-2 driven redox cycle intracellularly produces ROS that ultimately leads the organism to death. Therefore, understanding how these drugs interact with their target enzymes is crucial to develop more potent and safer agents for the treatment of TB infection.

We have undertaken extensive investigations to delineate the yet-unclear catalytic mechanism of NDH-2 and apply our knowledge to understand how NDH-2 inhibitors hinder the enzymatic reaction that is essential for survival of TB.

Figure 2. Structures of drugs that inhibit or interact with Mtb OxPhos, from left, phenothiazine (Thioridazine), Bedaquiline, and clofazimine.

Figure 3. Mechanism of action of clofazimine in the plasma membrane References: 1. Teh et al. (2007) Type II NADH: menaquinone oxidoreductase of Mycobacterium tuberculosis. Infect. Disord. Drug Targets 7: 169-81. 2. Weinstein et al. (2005) Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs. Proc. Natl. Acad. Sci.

USA 102: 4548-53. 3. Yano et al. (2006) Steady-state kinetics and inhibitory action of antitubercular phenothiazines on Mycobacterium tuberculosis type-II NADH-

menaquinone oxidoreductase (NDH-2). J. Biol. Chem. 281: 11456-63. 4. Warman et al. (2013) Antitubercular pharmacodynamics of phenothiazines. J. Antimicrob. Chemother. 68: 869-80. 5. Andries et al. (2005) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307: 223-7. 6. Yano et al. (2011) Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal

levels of reactive oxygen species. J. Biol. Chem. 286: 10276-87.

Screening of small molecule libraries for OxPhos inhibitors A high throughput screening (HTS) was carried out to identify compounds that inhibited the mycobacterium OxPhos. A novel membrane-based HTS assay was developed in our laboratory, the principle of which is described in Figure 6. The assay has produced robust dataset with z’ factor > 0.6 and can be applicable to other membrane systems.

Figure 6. Outline of ATP synthesis assay coupled with Luciferin/Luciferase system.

6. Acknowledgements • The authors thank Daniel Gentry, Andrew Ramsey, Trevor Selwood, and Jennifer Yano for discussion and Zenkun

Ma, Khisimuzi Mdluli, and Takushi Kaneko (TB Alliance) for their support and discussion. • We also thank Dr. Scott G. Franzblau (University of Illinois Chicago) for determining MIC against Mtb. • Instant JChem 5.9.2, 2012 and Marvin 5.5.1.0, 2011 from ChemAxon (http://www.chemaxon.com) were used for

structure database management, search, and prediction.

Mtb NDH-2 NADH-Q reductase reaction follows a ping-pong mechanism Kinetics of the Mtb NDH-2 NADH-Q reductase reaction was investigated using isolated and membrane-bound NDH-2 preparations with various exogenous Q substrates (UQ0, UQ1, UQ2, and menadione). The NADH-Q reductase activity of both soluble and membrane-bound NDH-2 showed a linear relationship measured at varying concentrations of NADH and Q at a constant concentration ratio ([Q] = αx[NADH], Figure 4). The linearity of the double reciprocal plots for each NDH-2 form is consistent with a ping-pong mechanism rather than a sequential mechanism that would exhibit a curved line. A B Figure 4. Kinetics for Q reduction with NADH by membrane-bound and purified Mtb NDH-2 activity. (A) Plots of initial velocities where both substrates were varied while maintaining a constant ratio of [UQ0] = 6.25 x [NADH]. (B) Ratio plots with purified NDH-2 were similar to that described in Figure 3A where [UQ1] = [UQ2] = 0.28 x [NADH] and [Menadione] = 0.68 x [NADH].

NADH and Q bind to different sites (Two-site ping-pong mechanism) The kinetic mechanism of the NDH-2 was further investigated by comparing the NADH-Q reductase reaction to the NDH-2 catalyzing NADH-thio-NAD+ (tNAD+) transhydrogenase reaction. The latter reaction follows a one-site ping-pong mechanism (Scheme 1). It was found that NDH-2 transhydrogenase activity was much slower (kcat ~ 0.25 sec-1) than the NADH-Q reductase activity (kcat ~ 500 sec-1) , suggesting that there is a rate-liming step in the hydride transfer reaction from FADH2 to tNAD+ (2nd half reaction) whereas NADH-Q reductase has a rate-limiting step in the 1st half reaction from NADH to FAD. Surprisingly, Km value for tNAD+ in the transhydrogenase reaction was 10 µM, indicating that tNAD+ has a high affinity to reduced NDH-2.

If NADH and Q share the same binding site as expected in a one-site ping-pong model, tNAD+ with the strong affinity to reduced NDH-2 is expected to compete with Q and strongly inhibits the NADH-Q reductase reaction. We compared tNAD+/NAD+ dead-end inhibition kinetics between NADH-Q reductase and transhydrogenase. The study revealed that tNAD+/NAD+ has much stronger affinity to reduced NDH-2 than oxidized form. However, tNAD+/NAD+ only weakly inhibits NADH-Q reductase. These results indicate that NAD(H) and Q bind to different sites. Considering the fact that no substrate inhibition was observed for NADH-Q reductase by either substrates, our study unambiguously concludes that Mtb catalyzes the NADH-Q reductase reaction by a two-site ping-pong mechanism (Scheme 2).

Mtb NDH-2 has two Q-binding sites Effects of quinols on NADH-Q reductase and transhydrogenase were studied. Unexpectedly, quinols showed only fractional inhibition for both reactions without affecting substrate affinities. However, in one instance with UQ0 as a substrate, the NADH-Q reductase activity was enhanced by UQ1H2 with a concomitant decrease in Km value for UQ0. These results indicate that there are two Q-binding sites in Mtb NDH-2: one is for substrate Q and the other shows the preferred affinity to quinol. The second Q-binding site appears to play a regulatory role. However, we cannot rule out a possibility that the second Q-site is involved in electron transfer from FADH2 to Q. Thus, exact roles of these two Q-binding sites remain to be determined in the future.

Eox Eox-NADH Ered-NAD+

NADH

Ered

tNAD+

Eox

tNADH NAD+/tNAD+

Eox-(t)NAD+

NAD+/tNAD+

Ered-tNAD+ Eox-tNADH Scheme 1 Strong

Weak

Scheme 2 Eox Eox-NADH Ered-NAD+

NADH

Ered

Q

Eox

QH2

NAD+/tNAD+

Eox-(t)NAD+

Strong

Ered-Q Eox-QH2 Weak

The results are consist with the crystal structures of yeast Ndi1 Recently, crystal structures of yeast ortholog Ndi1 were determined1,2. The structures by Feng et al.1 show that NAD(H) and Q bind to opposite faces of the FAD moiety consistent with a two-site ping-pong model (Figure 5A and B). The structure of Q-bound form revealed two Q molecules on the membrane binding surface (Figure 5C). One Q molecule (QI) is in a close vicinity to the FAD molecule with mutual interactions with the enzyme. The second Q molecule (QII) resides slightly away from the FAD but within a distance that allows electron transfer from either QI or FAD. Although structural and biochemical differences exist between yeast and Mtb NDH-2 enzymes, the catalytic mechanism and the enzymatic properties of Mtb NDH-2 determined in this study are in good agreement with the yeast structures. Figure 5. Crystal structures of yeast NDH-2 homolog. (A) NAD(H)-Q bound form, (B) NAD(H) and Q sites near the

FAD cofactor, and (C) two bound Q molecules in the Q bound form (reconstructed from Feng et al.1) Reference: 1. Feng et al. (2012) Structural insight into the type-II mitochondrial NADH dehydrogenases. Nature 491: 478-82. 2. Iwata et al. (2012) The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble

substrates. Proc. Natl. Acad. Sci. USA 109: 15247-52.

½O2 Cytochrome aa3 oxidase

ctaBCDE (Rv1451, Rv2200c, Rv3043c, Rv2193)

H2O

NADH:Q oxidoreductases

Type I: nuoA-N (Rv3145-Rv3158) Type II: ndh (Rv1854c) Type II: ndhA (Rv0392c)

Cytochrome bc1 complex

qcrABC (Rv2194-Rv2196)

Cyt c

Succinate:Q oxidoreductase

sdhABCD (Rv3316-Rv3319)

menABCDEG, ubiE (Rv0534c, Rv0548c, Rv0553, Rv0555, Rv0542c, Rv3853, Rv0558)

Menaquinone Pool

Cytochrome bd Oxidase

cydABCD (Rv1620c-1623c)

½O2

H2O

Generate ∆µH+ ⇒ ATP synthase

atpA-H (Rv1304-1351)

Figure 1. Oxidative phosphorylation system of M. tuberculosis.

NAD(H)

FAD QI

FAD

QI

QII Membrane surface

NAD(H)

FAD

Q

A B C

d[ATP] dt = 0

ADP + Pi ATP NADH

NAD+

PMF H+

Luciferin

Luciferase

Luminescence

H+ H+

½O2 H2O

ATP synthesis ≈ ATP consumption

Time

Lum

ines

cenc

e

Control

+Inhibitor

(1) Respiratory chain

(2) Membrane system

(3) ATP synthase

Periplasmic space

Cytoplasm

CFZred

CFZox

O2

ROS H2O

MQ

MQH2

½O2 + 2H+

Plasma membrane

H+

NAD+

NDH-2

NADH + H+

Cell Wall Skelton

NDH-2 inhibitors interact at the Q-binding sites Several NDH-2 specific inhibitors were indentified. Some compounds exhibit antimycobacterial activity. Physicochemical and biological properties are summarized in Table 1.

Table 1. Physicochemical and biological properties of the NDH-2 inhibitors *, MIC value against M. smegmatis. **, % inhibition at drug concentrations in parentheses.

Inhibition kinetics of the NDH-2 inhibitors were investigated. Compounds 1 and 2 competitively inhibited NADH-Q reductase with varying Q concentrations and fixed NADH concentration (Figure 7A). Compounds 3 and 4 showed an uncompetitive pattern with Q (Figure 7B) similar to phenothiazines reported earlier1. These inhibitors are uncompetitive with NADH. These compounds also inhibited ROS production that was mediated by CFZ. It is suggested, therefore, that the NDH-2 inhibitors and CFZ interact with the NDH-2 via the Q-binding site(s) that were revealed by our kinetic analysis.

Figure 7. Effects of NDH-2 inhibitors, Compound 1 (A) and Compound 4 (B), on NADH oxidation with UQ0 as a variable substrate and NADH at a fixed concentration (250 µM).

References: 1. Yano et al. (2006) Steady-state kinetics and inhibitory action of antitubercular phenothiazines on

Mycobacterium tuberculosis type-II NADH-menaquinone oxidoreductase (NDH-2). J. Biol. Chem. 281: 11456-63.

Compound MW cLogP

M. smeg ATP

synthesis IC50 (µM)

SMP ATP synthesis IC50 (µM)

Mtb NDH-2

inhibition Ki

MABA MIC (µM)

LORA MIC (µM)

Vero cytotox

(µM)

1 317 2.07 0.4 >100 4-5 nM n.d. n.d. n.d.

2 386 3.24 0.6 >100 0.5-2 µM 13* - 38

3 347 4.19 0.28 23.7 3-4 µM 15.2 40.7 >50

4 475 5.78 6.25 2.1 1-5 µM 81%**

(4.2 µM) 100%**

(42 µM) >12.6

• The present study revealed that Mtb NDH-2 catalyzes electron transfer from NADH to Q by a two-site ping-pong mechanism and that the enzyme contains two Q-binding sites with different affinities to Q substrates. The catalytic mechanism is consistent with the crystal structures of yeast NDH-2.

• The study demonstrated that the determined catalytic mechanism was highly valuable to understand the mechanism of inhibition by NDH-2 inhibitors that had been identified through HTS. It was shown that our NDH-2 inhibitors could be classified into two classes depending on their mode of inhibition. One class competes with Q for the substrate binding site. The other class exhibits an uncompetitive pattern with Q, indicating that the inhibitors bind to a site distinct from the substrate Q-binding site, possibly the second Q-binding site.

• CFZ-mediating ROS production via NDH-2 was inhibited by the NDH-2 inhibitors, suggesting that CFZ also binds to the Q-binding site(s). Further study is expected to define the interactions of CFZ with NDH-2 and will provide valuable information to design more potent compounds toward the treatment of TB infection.

A: Compound 1 B: Compound 4 0 nM

10 nM

20 nM

50 nM

100 nM

10 µM

0 µM

20 µM NAD+/tNAD+