Novel Antimycobacterial Compounds Suppress NAD Biogenesis by

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Novel antimycobacterial compounds suppress NAD biogenesis by targeting a unique pocket of NaMN adenylyltransferase Andrei L Osterman, Irina Rodionova, Xiaoqing Li, Edward Sergienko, ChenTing Ma, Antonino Catanzaro, Mark E. Pettigrove, Robert W Reed, Rashmi Gupta, Kyle H. Rohde, Konstantin V Korotkov, and LEONARDO SORCI ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00124 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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ACS Chemical Biology

1

Novel antimycobacterial compounds suppress NAD biogenesis by targeting

2

a unique pocket of NaMN adenylyltransferase

3 4

Andrei L. Osterman1, Irina Rodionova1,†, Xiaoqing Li1, Eduard Sergienko2, Chen-

5

Ting Ma2, Antonino Catanzaro3, Mark E. Pettigrove3, Robert W. Reed4,‡, Rashmi

6

Gupta5, Kyle H. Rohde5, Konstantin V. Korotkov4,*, and Leonardo Sorci6,*

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, United States 2 NCI Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, United States 3 Department of Medicine, University of California San Diego, La Jolla, CA, United States 4 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, United States 5 Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States 6 Department of Materials, Environmental Sciences and Urban Planning, Division of Bioinformatics and Biochemistry, Polytechnic University of Marche, Italy †

present address: Department of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States ‡ present address: Division of Regulatory Services, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, United States * Correspondence to: Leonardo Sorci, [email protected]; Konstantin V. Korotkov, [email protected].

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ABSTRACT

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Conventional treatments to combat the tuberculosis (TB) epidemic are falling short,

33

thus encouraging the search for novel antitubercular drugs acting on unexplored

34

molecular targets.

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bioactive compounds with potent antitubercular activity. However, their cellular

36

target and mechanism of action remain largely unknown. Further evaluation of

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these compounds may include their screening in search for known antitubercular

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drug targets hits. Here, a collection of nearly 1,400 mycobactericidal compounds

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was screened against Mycobacterium tuberculosis NaMN adenylyltransferase

40

(MtNadD), a key enzyme in the biogenesis of NAD cofactor that was recently

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validated as a new drug target for dormant and active tuberculosis. We found three

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chemotypes that efficiently inhibit MtNadD in the low micromolar range in vitro.

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SAR and cheminformatics studies of commercially available analogs point to a

44

series of benzimidazolium derivatives, here named N2, with bactericidal activity on

45

different

46

tuberculosis, and dormant M. smegmatis. The on-target activity was supported by

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the increased resistance of an M. smegmatis strain overexpressing the target and

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by a rapid decline in NAD(H) levels. A co-crystal structure of MtNadD with N2-8

49

inhibitor reveals that the binding of the inhibitor induced the formation of a new

50

quaternary structure, a dimer-of-dimers where two copies of the inhibitor occupy

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symmetrical positions in the dimer interface, thus paving the way for the

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development of a new generation of selective MtNadD bioactive inhibitors. All

Several whole cell phenotypic screenings have delivered

mycobacteria,

including

M.

abscessus,


multidrug-resistant

M.

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these results strongly suggest that pharmacological inhibition of MtNadD is an

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effective strategy to combat dormant and resistant Mtb strains.

55 56

INTRODUCTION

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Despite extensive prevention and control measures in the last two decades,

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Tuberculosis yet represents a dramatic health issue with global impact. The World

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Health Organization (WHO) estimates over ten million active disease cases in

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2016

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immunocompromised HIV-positive individuals1. Mycobacterium tuberculosis

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(Mtb), which causes TB, undergoes a metabolic shift in the human host into a

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dormant state resulting in long-term persistent infection and phenotypic resistance

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to many otherwise successful drugs2-4. Drug-susceptible strains can be effectively

66

treated with a 6 to 9-month regimen of multiple antibiotics, but the non-adherence

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to the therapeutic guidelines on a global level has exacerbated the selection and

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spreading of resistant strains in the last two decades5. Particularly alarming is the

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increase of multidrug-resistant tuberculosis (MDR-TB), resistant to isoniazid and

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rifampicin, two of the most powerful TB drugs, and extensively drug-resistant

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(XDR-TB), which are additionally resistant to any fluoroquinolone, and to at least

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one of the second line drugs (amikacin, capreomycin or kanamycin)6.

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Consensus is growing that novel, much-needed antibiotics, should efficiently kill

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drug-resistant strains and dormant mycobacteria as well as shorten therapy via

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novel targets2, 3.

and

nearly

1.7

million

deaths,

22%


of

which

were

among


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Two novel classes of antibiotics, diarylquinolines and nitroimidazoles, have

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reached stage III of clinical trials, proving to be effective alone in treating latent

78

tuberculosis (LTB)

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diarylquinolines, is the first new drug in decades to be clinically approved for TB

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treatment. In particular, due to its unique mechanism of action on mycobacterial

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ATP synthase, bedaquiline opened a new door for discovering anti-TB/LTB drugs

82

using oxidative phosphorylation as a target system11. However, resistance to BDQ

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has already been observed. Besides the selection of mutations of the atpE subunit

84

of target ATP synthase, drug-responsive mechanisms involve activation of

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dormancy and metabolic remodeling

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from substrate-level phosphorylation appears to enable transient bacterial survival,

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as further confirmed by BDQ-enhanced killing of mycobacteria on non-fermentable

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energy sources13. Glycolysis not only produces ATP faster, albeit less efficiently,

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than oxidative phosphorylation but also supplies metabolic precursors required for

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macromolecular biosynthesis. Strikingly, glycolytic enzymes are upregulated in

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BDQ-treated mycobacteria, including lactate dehydrogenase that recycles NADH

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to NAD13.

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aspects of energy metabolism that are essential for both actively replicating and

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dormant forms of mycobacteria.

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Biogenesis and homeostasis of redox cofactors, most notably of NAD pool

96

(NAD(H) and NADP(H)), is another crucial aspect of respiratory energy

97

metabolism in both, replicating and dormant forms of mycobacteria 14, 15.

7-10.

Bedaquiline (BDQ), the first-in-class compound of

12, 13.

Notably, alternative ATP production

These observations emphasize the significance of targeting other


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NAD(H) is the entry electron donor in the respiratory chain and the key oxidant

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driving the glycolysis. Thus, depletion of NAD(H) pool generates a glycolytic

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slowdown

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synthesis from both sources.

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In this view, NAD starvation, which represents alone a validated strategy to kill

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mycobacteria recalcitrant to current therapies17, 18, may potentially show synergy

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with drugs targeting ATP synthesis such as bedaquiline.

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The universally conserved and essential enzymes, NadD, NadE, and NadF, which

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drive the last non-redundant steps of NAD(P) biosynthesis, were implicated by our

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previous genomics-driven studies as potential drug targets in numerous bacterial

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pathogens19-22. Of these three enzymes, NadD is the most divergent from its

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human counterparts (NMNAT1-323), offering the opportunity of developing

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selective small-molecule inhibitors22, 24-27. Our recent studies showed that induced

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degradation of NadD and NadE enzymes in a model system of M. smegmatis

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(Msm) led to rapid depletion of the NAD cofactor pool followed by cell death, even

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under non-replicative conditions17. Another group used a similar approach to show

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that induction of NadE degradation also suppressed acute and chronic Mtb

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infection in mice18. These findings provided the ultimate validation of NAD

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metabolism as a target pathway for the development of new antimycobacterial

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therapies. Structure-function and inhibition studies of the key NAD(P) biosynthetic

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enzymes 22, 24, 25, 28, 29 including our recently published work on MtNadD26 support

119

their druggability.

16

and rapid shutdown of electron transfer chain thereby affecting ATP



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We set out to identify inhibitors of MtNadD through a target-based screen

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of orphan compounds with mycobactericidal activity. Here, we report the

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identification as well as the biochemical and biological characterization of several

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potent inhibitors of NadD with bactericidal activity on mycobacteria, including M.

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abscessus, multidrug-resistant M. tuberculosis, as well as a prominent bactericidal

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effect under non-replicating conditions in M. smegmatis. To clarify in atomic detail

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the mode of action of these compounds and their structure-activity relationships,

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we also determined the crystal structure of MtNadD in complex with one of these

128

inhibitors.

129 130

RESULTS AND DISCUSSION

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Screening of bioactive antimycobacterial compounds affords novel MtNadD

133

inhibitors. Previous whole cell phenotypic screening has identified potent anti-

134

mycobacterial compounds in a variety of tested conditions on replicating, non-

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replicating and intracellular forms of Mtb. These compounds were selected via

136

several cell-based HTS campaigns as reported in PubChem30,

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mechanisms and molecular targets remain unknown. We speculated that, given a

138

limited space of potential targets (~200–300 essential genes in M. tuberculosis),

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at least some of these compounds could target the essential enzymes of NAD

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metabolism. For this reason, a collection of 1389 bioactive compounds, kindly

141

provided by Global Alliance for TB Drug Development (TB Alliance) was tested

142

against the recombinant M. tuberculosis NadD enzyme using colorimetric 6 ACS Paragon Plus Environment

31,

but their

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143

detection assay of released PPi (Figure 1). The primary assays, performed at a

144

fixed final concentration of 25 µM for each compound, and at subsaturating

145

concentrations of ATP and NaMN, identified 16 compounds (~1% hit rate)

146

inhibiting more than 30% of enzymatic activity, with three of these demonstrating

147

>50% inhibition. Notably, this hit rate is comparable to our previous structure-

148

based approach on NadD from Escherichia coli and Staphylococcus aureus

149

which, however, used a more stringent concentration of the inhibitor (100 µM).

150

These primary hits were repurchased from different vendors, and their

151

concentration-response profiling yielded 5 compounds with IC50 values in the

152

range of 6–22 µM (Supplementary Table 1). The three top-ranked compounds with

153

significant structural diversity, termed here as N1, N2, and N3, were selected for

154

further analysis, including cheminformatics and SAR studies by analogs’ testing,

155

cell-based assays, and co-crystallization trials (Figure 1).

24

156 157

SAR for chemotypes N1, N2, and N3. Before any further investigation on these

158

chemotypes was pursued, we wanted to confirm their bioactivity as initially

159

displayed in whole cell phenotypic assay. For example, we determined a minimal

160

inhibitory concentration of 28 µM of the compound N2-11 for M. tuberculosis

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H37Rv. This result is consistent with the inhibition value reported in PubChem

162

Bioassay 1626 (MIC of 16 µM). Overall, around 30 analogs of the three selected

163

chemotypes were purchased from different vendors and tested in vitro against

164

MtNadD (Figure 2 and Tables 1–3). Despite a limited chemical search exploration,

165

four analogs show improved MtNadD inhibition relative to their parent compound, 7 ACS Paragon Plus Environment


166

with up to 5-fold lower IC50 (2.5 µM) for N1-11 analog, which represents the most

167

potent inhibitor for MtNadD described so far 26.

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In class N1, the replacement of a 6-fluoro benzyl group with the more

169

hydrophobic phenylethyl (N1-11) or benzyl (N2-11) moieties yielded the best

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improvement. More polar moieties such as five or six-membered rings with two or

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more heteroatoms were poorly tolerated (IC50>50–100 µM), while linear or

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branched hydrocarbon chains (N1-6 through N1-8), or the smaller cyclopropyl

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moieties (N1-9) were well-tolerated substitutions (15 µM 100 >50 >50 >50

54.81 54.28 79.9 97.0 22.14 11.37 14.68 – – 20.43 – – 38.2 – – 41.76 – – – 14.6 – – – – –

Mycobacterium Growth inhibition (%) at MIC (µM) 50 µM tuberculosis smegmatisc e d


– – – – – nd nd ~50 ~20 nd ~50 nd nd nd nd ~20 nd ~50 ~100 ~10 ~20 nd nd nd nd

– – – – – – – – – 100 – – – – – – – – –

>25 50 25 25 >25 nd >25 – – 25 – – 25 – – nd – – – nd – – – – –

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N1-26 N1-27 N1-28 N1-29 N1-30 N1-31

1423254 1897037 3774654 658395 16682092 3333039

>50 >100 >100 – >100 –

– – – 19.8 22.17 54.21

nd nd nd – ~20 –

– – – >10 >100 –

– – – 25

447 448

a

449

Standard errors are reported. b in vitro inhibition of M. tuberculosis H37Rv fructose-

450

bisphosphate aldolase (FBA), as reported in PubChem Bioassay AID 588726.

451

Antibacterial activity against M. smegmatis is expressed as a percentage of growth

452

suppression at fixed 50 µM concentration, except for N1-18 that was tested at 20

453

µM.

454

obtained from PubChem Bioassay AID 449762 data. “ – “ , not assayed; nd, no

455

detected effect. In red, compounds identified in the primary screening. In blue,

456

analogs identified via cheminformatics search for which bioactivity is reported, but

457

not tested in our study.

Assays performed in duplicate in at least five different inhibitors concentration.

d

values are deduced from PubChem Bioassay AID 1626 data.

e

c

values

458 459

Table 2. Inhibitory properties of N2 class MtNadD inhibitors evaluated

460 ID

PubChem CID

MtNadDa IC50 (µM)

N2-1 N2-2 N2-3 N2-4 N2-5 N2-6 N2-7 N2-8 N2-9

2834410 2834412 2834423 2834427 2834425 2834414 2834416 2834419 16192954

5±1 111±16 – – – 46±16 13±3 18±2 18±3

Mycobacterium Growth inhibition, MIC (µM) tuberculosis smegmatis abscessus b c 5 25 – – – 25 5 5 5

46.7 – – – – I – 32.7 – I – I – 16.6 21.2 I 38.5 10.4


– – I 28 I I – I I

61.4 – – – – – – 26.7 26.2


461

N2-10 N2-11

3908383 16195070

20±3 6±1

5 5

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19.7 – 28.2 15.9

– I

25.6 26

462

aAssays

463

Standard errors are reported. “–“ , not assayed; nd, no detected effect; b,c, values

464

deduced from PubChem Bioassay AID 1626 and AID 449762 data, respectively.

465

For consistency, these values represent the minimal inhibitory concentration

466

yielding >99% growth suppression and were obtained by fitting the reported EC50

467

and Hill Slope values into ECanything built-in equation in Prism 7.0. I, compounds

468

tagged as “Inconclusive” and which showed >98% inhibition at a single

469

concentration of 25 µM”. No dose-response analysis was carried out. ID color-

470

coding is as in Table 1.

performed in duplicate in at least five different inhibitors concentration.

471 472

Table 3. Inhibitory properties of N3 class MtNadD inhibitors. ID

473

N3-1 N3-2 N3-3

MtNadD IC50 (µM) 11±1 96±26 >100

Growth inhibition, MIC (µM) M. smegmatis

M. tuberculosis

M. abscessus

10 >50 nd

3.1 >100 –

20.9 >100 –

474

Assays performed in duplicate in at least five different inhibitors concentration.

475

Standard errors are reported. “ – “ , not assayed; nd, no detected effect (at 50

476

µM). ID color- coding is as in Table 1 and 2.

477

Table 4. Data collection and refinement statistics. M. tuberculosis NadD in complex with N2-8 (PDB 6BUV) Data collection Wavelength (Å)

1.0000 22 ACS Paragon Plus Environment

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Space group Cell dimensions: a, b, c (Å)    () Resolution (Å) Rsym CC1/2b I / I Completeness (%) Multiplicity Refinement Resolution (Å) No. reflections (total / free) Rwork / Rfree Number of atoms: Protein Ligand/ion Water B-factors: Protein Ligand/ion Water All atoms Wilson B R.m.s. deviations: Bond lengths (Å) Bond angles () Ramachandran distributionc (%): Favored Allowed Outliers Rotamer outliersc (%) Clashscored MolProbity scoree

H32 163.73, 163.73, 153.65 90, 90, 120 52.1–1.86 (1.91–1.86)a 0.077 (1.146) 99.6 (54.7) 11.08 (1.72) 99.3 (100.0) 3.8 (3.8) 52.1–1.86 65711 / 5923 0.172 / 0.192 2976 38 296 43.7 60.7 49.6 44.4 33.4 0.008 1.008 98.9 1.1 0 0 2.53 1.04

478 479

aValues

480

bCC

481

by XSCALE 44.

482

cCalculated

483

dClashscore

1/2

in parentheses are for the highest-resolution shell.

correlation coefficient as defined in Karplus & Diederichs 43 and calculated

using the MolProbity server (http://molprobity.biochem.duke.edu) 45. is the number of serious steric overlaps (> 0.4 Å) per 1000 atoms. 23 ACS Paragon Plus Environment


484

eMolProbity

485

evaluations into a single score, normalized to be on the same scale as X-ray

486

resolution 45.

score combines the clashscore, rotamer, and Ramachandran

487 488

FIGURE LEGENDS

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Figure 1. Flowchart of the whole-cell and target screening approach for

490

delivering novel antitubercular NaMN adenylyltransferase inhibitors.

491 492

Figure 2. Chemical diversity of the three series of novel MtNadD inhibitors

493

evaluated.

494

In red, molecules that were identified in our primary screen of the TB Alliance

495

bioactive library against MtNadD (IC50 < 20 µM). In black, novel analogs for each

496

chemotype tested in this study. In blue, analogs that were not tested in our study,

497

but for which a relevant activity against M. tuberculosis or an Mtb target enzyme

498

has been reported in PubChem (see Tables 1–2 and main text for details).

499 500

Figure 3. Bactericidal, on-target effects of N2 inhibitors in replicating and

501

non-replicating M. smegmatis.

502

(A–C) Growth suppression curves at different N2 series inhibitors’ concentrations

503

underline their killing activity on replicating Msm at above 5 µM. DMSO at 1 %

504

represents the “no inhibitor” control. Linezolid at 3 µM was used as a positive

505

control. (D) Addition of the inhibitor at ~ 5xMIC concentration at log-phase (after

506

12.5 hours of growth) stops the growth of Msm and (E) rapidly deplete NAD pool.


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507

Residual levels of NAD were measured by a colorimetric detection kit and

508

normalized by total protein in samples taken for 6 hours, in 2 hours’ time interval.

509

(F) Cell viability by CFU assay after 1–4 days of incubation of Msm with 5xMIC

510

inhibitor (25 µM) under carbon starvation conditions. In this case, Linezolid was

511

used at 15 µM.

512 513

Figure 4. Structure of MtNadD in complex with inhibitor N2-8.

514

(A) N2-8 inhibitor-driven dimer-of-dimers of MtNadD with two molecules of the

515

ligand bridging the dimers formed by chains B and A’, and chains B’ and A,

516

respectively. (B) A close-up view of the N2-8 binding site. The hydrophobic

517

residues lining up the inhibitor binding site are shown in sticks representation. A-

518

weighted 2FO–FC electron density map contoured at 1 is shown as blue mesh.

519

(C) Comparison of the closed active-site conformation of MtNadD•N2-8 complex

520

(light blue) with MtNadD apo structure (yellow) (PDB ID, 4X0E) and

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MaNadD•NaAD complex structure (purple) (PDB ID, 5DEO). H-bonding between

522

D109 and the adenosine moiety of the MaNadD•NaAD complex is shown as

523

dashed line. The steric clashes of L164 with adenine ring, I113 with nicotinate ring,

524

and G106 with the AMP ribose are marked by a dotted circle.

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Figure 1. Flowchart of the whole-cell and target screening approach for delivering novel antitubercular NaMN adenylyltransferase inhibitors. 121x160mm (300 x 300 DPI)

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Figure 2. Chemical diversity of the 3 series of novel MtNadD inhibitors evaluated. In red, molecules that were identified in our primary screen of the TB Alliance bioactive library against MtNadD (IC50< 20 μM). In black, novel analogues for each chemotype tested in this study. In blue, analogs that were not tested in our study, but for which a relevant activity against M. tuberculosis or a Mtb target enzyme has been reported in PubChem (see Tables 1-2 and main text for details). 134x163mm (300 x 300 DPI)


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Figure 3. Bactericidal, on-target effects of N2 inhibitors in replicating and non-replicating M. smegmatis. (A-C) Growth suppression curves at different N2 series inhibitors’ concentrations underline their killing activity on replicating Msm at above 5 μM. DMSO at 1 % represents the “no inhibitor” control. Linezolid at 3 μM was used as a positive control. (D) Addition of the inhibitor at ~ 5xMIC concentration at log-phase (after 12.5 hours of growth) stops the growth of Msm and (E) rapidly deplete NAD pool. Residual levels of NAD were measured by a colorimetric detection kit and normalized by total protein in samples taken for 6 hours, in 2 hours’ time interval. (F) Cell viability by CFU assay after 1-4 days of incubation of Msm with 5xMIC inhibitor (25 μM) under carbon starvation conditions. In this case, Linezolid was used at 15 μM. 134x116mm (300 x 300 DPI)



Figure 4. Structure of MtNadD in complex with inhibitor N2-8. (A) N2-8 inhibitor-driven dimer-ofdimers of MtNadD with two molecules of the ligand bridging the dimers formed by chains B and A’, and chains B’ and A, respectively. (B) A close-up view of the N2-8 binding site. The hydrophobic residues lining up the inhibitor binding site are shown in sticks representation. σA-weighted 2FO–FC electron density map contoured at 1σ is shown as blue mesh. (C) Comparison of the closed active-site conformation of MtNadD•N2-8 complex (light blue) with MtNadD apo structure (yellow) (PDB ID, 4X0E) and MaNadD•NaAD complex structure (purple) (PDB ID, 5DEO). H-bonding between D109 and the adenosine moiety of the MaNadD•NaAD complex is shown as dashed line. The steric clashes of L164 with adenine ring, I113 with nicotinate ring, and G106 with the AMP ribose are marked by a dotted circle. 177x195mm (300 x 300 DPI)


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Novel Antimycobacterial Compounds Suppress NAD Biogenesis by

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