PufA inhibitors discovery

1. Discovery and Mechanistic Study of Mycobacterium tuberculosis PafA Inhibitors
Journal of Medicinal Chemistry; August 04, 2022, 65, 11058−11065​
Chemistry, Medicinal; 3/63 = 4.76%; IF: 8.039
SHUAIB AHMAD (R. Ph)
Pharm.D, M.Phil. Pharmacology (ICCBS, PCMD - Pakistan)
Ph.D. (Count.) School of Pharmacy,
Kaohsiung Medical University - Taiwan
Advisor: Prof. Dr. Ying-Chi Lin
Email: shuaib.pcmd@iccs.edu

2. CONTENTS
INTRODUCTION
AIMS OF THE STUDY
METHODOLOGY
RESULTS
CONCLUSIONS
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3. INTRODUCTION
 The causative agent of Tuberculosis infection is the bacterium Mycobacterium tuberculosis.
 Tuberculosis caused 1.5 million deaths in 2020 and is the second leading infectious killer after COVID-19 (WHO, 2020).
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4. Global Trends in the Estimated Number of Tuberculosis
WHO global TB reports from 1990 to 2021. Adapted from the
World Bank database
Past and future trajectories of TB incidence in three high-burden countries
Cases and deaths and Tuberculosis Mortality, 2000 to 2020.
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5. TB Infection
• Latent TB (LTB) infection is a state of persistent immune response to the activation by Mycobacterium tuberculosis antigens without evidence of clinically manifested active TB as a result
of immunological control.
• Active pulmonary TB disease either appears within 1–2 years after infection or following reactivation of latent infection with progressive bacterial replication and pulmonary necrosis and
often but not always includes cavitary lesions that promote the transmission of bacteria.
• Cavitary TB is an active disease characterized by the presence of open pulmonary cavities in the lung parenchyma, composed of a wall filled with necrotic debris and high bacterial burden,
leading to poor treatment outcomes and increased person-to-person transmission.
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6.  MDR/RR and XDR TB are major public health crises (WHO, 2020).
 There is an urgent need to develop new treatment options for tuberculosis with a unique mechanism of action and specific to Mtb.
 One attractive target is proteasome accessory factor A (PafA), the only ligase to designate proteins for degradation in Mtb.
 Delamanid was approved by the EMA only, and pretomanid was approved by the FDA for use in the bedaquiline–pretomanid–linezolid regimen.
Anti-Tuberculosis Drug Candidate Pipeline
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7. Mechanism of Drug Action
 The majority of novel targets for anti-tuberculosis agents are membrane-associated.
 The bedaquiline, TBAJ-876 and TBAJ-587 target the ATP synthase.
 The nitroimidazoles pretomanid and delamanid exhibit a dual mode of action under low and normal oxygen tension, poison multiple essential pathways, and are bactericidal against
replicating and non-replicating mycobacteria.
 SQ109 and the MPL series are the most advanced among a broad panel of agents targeting MmpL3, involved in the export of trehalose monomycolate, a mycolic acid component.
 Three chemically distinct series all target DprE: OPC167832, TBA7371, and BTZ043.
 GSK656 is the first oxaborole in clinical development targeting a mycobacterial tRNA synthetase.
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9. 8
Drug Targets

10. Pupylation Reaction in Mtb
Like the ubiquitination-degradation system in eukaryotes, there is also a protein degradation system in Actinobacteria and Nitrospira phyla bacteria such as Mtb and
Mycobacterium smegmatis.
In Mtb, PafA fixes to a 64 amino acid prokaryotic ubiquitin-like protein (Pup) to a protein and the bacterial proteasome destroys that protein.
In the mycobacterial pupylation pathway, the C-terminal glutamine of Pup is first deamidated by deamidase of Pup (Dop), after which Pup is coupled by proteasome accessory factor A
(PafA) to the ε-amino group of a substrate lysine via an isopeptide bond.
Pupylation activity is a tightly regulated process, so inhibiting and activating PafA may disrupt bacterial protein homeostasis, thereby inhibiting Mtb.
Pupylation pathway in mycobacteria
Modes of coupling: (I) C-terminal carboxylate; (II) Carboxylate of the glutamate side chain
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11. AIMS OF THE STUDY
r storing and retrieving new information.
• The
1. To develop a plate-based high-throughput screening assay and screen a library of drug-like compounds.
2. To identify inhibitors or activators of PafA.
3. The inhibition mechanisms and antiproliferative activity of the PafA inhibitors.
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12. METHODOLOGY
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1. Cloning, Protein Expression, and Purification:
 GFP-Pup (39- 64, Q64E, Mtb), Mtb PanB, Mtb InhA, Mtb KasA, Mtb DHFR, Mtb PafA, and its mutants were cloned into a pET-28a+ expression vector incorporating an N-terminal
6× His-tag.
 Mtb PknB was cloned into an H6MBP-Psyno-1 expression vector incorporating a TEV-cleavable N-terminal MBP-tag.
 Mtb Pup (39-64, Q64E) was cloned into a pGEX- 4T1 expression vector incorporating an N-terminal GST tag.
 An E48R mutation was introduced into GST-Pup to enhance the binding affinity to PafA. Mtb Pup (1-64, Q64E) was cloned into a pET-15b+ expression vector incorporating an N-terminal
6× His-tag.
 The plasmid was transformed into E. coli (BL-21) and grown in a liquid broth medium. After adding 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG), the protein was expressed for
3h at 37 °C. Cells were harvested and sonicated in lysis buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol.
 Proteins were purified by the respective purification tags, followed by Q-sepharose anion exchange chromatography.
2. Plate-Based High-Throughput Screening Assay

13. 3. In Solution Pupylation Assay
 Substrates, PafA, and GFP-Pup (39-64, Q64E) were incubated in a reaction buffer containing 50 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM DTT, 5 mM MgCl2, and 3 mM ATP
at room temperature overnight.
 The resulting samples were separated by SDS-PAGE and visualized by Coomassie blue staining.
4. GST Pull-Down Assay
 GST and GST-pup were immobilized on GSH beads. Soluble proteins (1 μM) and compounds (20 μM) were incubated with the immobilized proteins in a total volume of 500 μL at 4 °C for
1.5 h.
 After two washing steps, bound proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining.
 Pull-down buffer contained 20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM MgCl2, and 0.005% Triton X-100.
5. Differential Scanning Fluorescence
 PafA (10 μM) was incubated with 50 μM different compounds or DMSO buffer for 0.5 h in 0.2 mL strip tubes.
 The assay buffer contained 30 mM Tris pH 8.0, 150 mM NaCl, and 5 mM MgCl2.
 After incubation, 5X SYPRO Orange (Thermo Fisher) was added to the samples. The samples were heated in a CFX96 Real-time System (BioRad) from 25 to 95 °C in 0.5 °C steps.
 The data were processed with CFX Manager software and presented with GraphPad
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14. 6. Molecular Docking
 Docking was carried out using the LibDock portion of Discovery Studio 3.1.
 The protein structure of mtPafA was obtained from the AlphaFold protein structure database (ID: A5U4C3) and prepared by adding hydrogens.
 The chemical structure of ST1926 and bithionol was prepared and optimized before docking.
 The final images were prepared by PyMOL (http://pymol.org)
7. M. tuberculosis H37Ra MIC Assay
 M. tuberculosis H37Ra was cultured in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 10% v/v oleic acid albumin dextrose catalase (OADC), and 0.2% v/v glycerol.
 Two fold serially diluted compounds (starting at 200 μM) were added to each well of H37Ra culture (106 CFU/mL) in 96-well microplates.
 The plates were incubated for 7 days at 37 °C. Each well was incubated with 20 μL of 0.01% alamar Blue and 12.5 μL of 20% Tween 80 for 24 h.
 The fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm by a microplate reader 3550-UV (Thermo Fisher).
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15. RESULTS
Designing a More Stable and Easier-to-Purify Mtb PafA Protein
 To screen for M. tuberculosis PafA (mtPafA) inhibitors or activators, a large quantity of active mtPafA was used.
 PROSS-suggested mutation, V85I. The mutation fills a small void volume inside the protein (shown is a homology-modeled mtPafA structure) to enhance protein stability.
 Purification of the mtPafA mutant (mtPafA*). The mutant was expressed at 37 °C. Analyzed were the supernatant (S), the resuspended insoluble pellet (P) after cell lysis and centrifugation,
the impurities washed off (W), and the Ni-NTA Sepharose beads bound contents after washing (B).
 Pupylation activity of mtPafA (0.1 μM) and cgPafA (0.1 μM) at different time points at 37 °C.
 Pup, PanB, and ATP were used at 4 μM, 2 μM, and 3 mM, respectively. Pup was labeled with the fluorophore 5-iodoacetamidofluorescein (IAF) to enhance the detection efficiency.
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16. Establishing a High-Throughput Screening Assay
 PanB was first immobilized onto polystyrene plates in NaHCO3 buffer.
 GFP-tagged Pup, PafA and ATP were then added to the wells and incubated to allow the reaction to occur.
 After incubation, the wells were washed, and GFP fluorescence was detected with a plate reader.
 A time-dependent increase in GFP fluorescence was observed for reactions in the presence of ATP.
 The assay was further validated with the reported PafA inhibitor AEBSF, which showed that 2 mM AEBSF dramatically reduced the level of GFP fluorescence.
39-64, Q64E
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17. Identification of Three Pupylation Modulators
 Screened 1354 drug-like compounds library.
 Through the screen, two inhibitors (E-H8, V-G3) and one activator (B-F2) were identified.
 On further analysis, it was found that E-H8 was selective against mtPafA, but V-G3 inhibited both mtPafA and cgPafA.
 IC50 values for E-H8 and V-G3 against mtPafA were measured to be 17.9 and 15.9 μM, respectively.
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E-H8 V-G3
17.9 μM
15.9 μM

18.  ST1926 (E-H8) is an atypical retinoid that has been reported to induce DNA damage and cell apoptosis in tumor cells
ST1926 at 20 μM did not work through binding to PanB, since it additionally inhibited the pupylation of several Mtb proteins including KasA, InhA, PknB, and DHFR.
 A differential scanning fluorescence (DSF) assay showed that ST1926 increased the Tm of PafA by 6.5 °C.
 The GST-pup pull-down result showed that ST1926 disrupted the interaction between PafA and Pup, showing that ST1926 was likely bound in the Pup-binding groove.
E-H8 Inhibits PafA by Binding to a Specific Site in the Pup-Binding Groove
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ST1926

19.  Since ST1926 inhibited mtPafA but not cgPafA, it was reasoned that ST1926 bound in a particular region of the Pup-binding groove that is different between the two species.
 ST1926 carries a negative charge, the difference in electrostatic surface potential around 19H and G20 may be responsible for ST1926 selectivity.
 To confirm the ST1926-binding location, mtPafA residues 19H, G20 together with the surrounding F18 and H21, were mutated to the cgPafA equivalent residues DGDS.
 The mt*swap construct contains DGDS instead of FHGH and the cg-swap construct contains FHGH instead of DGDS, showing reverse selectivity towards these two PafA enzymes.
E-H8 Inhibits PafA by Binding to a Specific Site in the Pup-Binding Groove
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cgPafA mtPafA ST1926

20. ST1926
 The docked poise of ST1926 (blue spheres) in the Pup-binding groove of PafA. The two panels are related to each other by a 90° rotation.
 The adamantyl of ST1926 invariably docked into a hydrophobic pocket formed by F109, Y107, T17, Q352, and L355.
 This pocket is occupied by the F54 of Pup in the complex structure of PafA-Pup.
 The carboxyl of ST1926 hydrogen-bonded with the V14 main chain, as the Q60 of Pup.
 Inhibition (MIC 90%) with the value of 164 μM against the H37Ra strain of Mtb was obtained for ST1926.
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E-H8 Inhibits PafA by Binding to a Specific Site in the Pup-Binding Groove

21. V-G3 Inhibits Pupylation by Binding to the ATP Binding Pocket
 V-G3 (bithionol) is a clinically approved antiparasitic drug that also has antitumor and antibiotic activities against Staphylococcus aureus.
 Bithionol also inhibited the pupylation of several Mtb endogenous proteins.
 Bithionol increased the Tm of PafA by 2.5 °C, suggesting that it inhibited pupylation by binding to PafA.
 It was found that changing the concentration of Pup did not affect the inhibition potency of bithionol, but changing the concentration of ATP and PafA did.
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Bithionol

22. V-G3 Inhibits Pupylation by Binding to the ATP Binding Pocket
 The results indicated that bithionol is likely bound in the ATP-binding pocket of PafA.
 Bithionol inhibited both cgPafA and mtPafA .
 Bithionol showed good shape complementation to the ATP-binding pocket, forming many hydrophobic interactions in addition to one hydrogen bond to the carbonyl oxygen of T66.
 In H37Ra strain of Mtb, bithionol showed an MIC90 value of 89.9 μM.
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PafA
mtPafA
cgPafA

23.  For finding structure-activity relationships, 16 bithionol derivatives with different substitutions were synthesized.
 Results showed that compounds lacking a hydroxyl substitution displayed no or very weak PafA inhibition.
 The hydroxyl-containing compounds, 17, 18, and 20 did not inhibit PafA.
 R4 is a hydroxyl group and both R1 and R6 are non-hydrogen atom substituents, the compounds have better effects on PafA binding.
 Docking of potent derivatives, 16 and 19, into PafA confirmed that the hydroxyl groups were indeed hydrogen bonded with T66 carbonyl.
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V-G3 Inhibits Pupylation by Binding to the ATP Binding Pocket
PafA
Chemical Structures of V-G3 Derivatives

24. B-F2 Promotes Pupylation of mtPafA but Slightly Inhibits that of cgPafA
 B-F2 (YM155, sepantronium bromide) inhibits survivin protein expression and the proliferation of several cancers.
 B-F2 also enhanced the pupylation of these Mtb proteins in solution, concomitantly depleting 5IAF-Pup.
 Intriguingly, B-F2 slightly inhibited cgPafA but did not activate.
 The EC50 values for pupylation of PanB and KasA were 1.7 and 2.0 μM, respectively.
 In the attenuated H37Ra strain of Mtb, B-F2 exhibited an MIC90 value of 6.7 μM.
 The activation mechanism of B-F2 is under investigation.
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The pupylation of different Mtb proteins by cgPafA
Inhibition of Mtb H37Ra

25. CONCLUSIONS
 In this work, a more stable mutant with higher expression of mtPafA was designed, which was essential for the success of later screening and mechanistic investigation and required a
large number of stable proteins.
 mtPafA and cgPafA were found to be considerably different since ST1926 showed selectivity between mtPafA and cgPafA and since BF-2 (YM155) was a mtPafA activator but a cgPafA
inhibitor.
 In future screening with larger compound libraries, compounds can be screened at lower concentrations (preferably less than 1 μM).
 In previous reports, AEBSF inhibits PafA at millimolar concentrations and several inhibitors inhibit PafA at 100 μM.
 In this work, the author identified two micromolar range inhibitors, ST1926 and bithionol.
 Bithionol was slightly more potent than ST1926 in the E. coli endogenous protein pupylation assay, although they were similarly potent in enzyme assays.
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