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. 2026 Sep 17;11(38):57583–57594. doi: 10.1021/acsomega.6c04844

Atomic-Level Structures Reveal How a Repurposed 3‑Aminopyrazine-2-carboxamide Scaffold Inhibits Mycobacterium tuberculosis Prolyl-tRNA Synthetase

Priyanka Gade †,‡, Karolina Michalska †,‡, Jan Zitko §, Jacek Wower ∥, Natalia Maltseva †, Vinod Sukanth Kumar Pallabothula §, Robert Jedrzejczak †, Andrzej Joachimiak †,‡,⊥,*
PMCID: PMC13629369  PMID: 42825133

Abstract

Tuberculosis (TB) is again the world’s leading cause of death from a single infectious agent. Aminoacyl-tRNA synthetases are essential for protein synthesis and are promising drug targets because bacterial and human enzymes differ. We repurposed a human prolyl-tRNA synthetase (ProRS) inhibitor scaffold, 3-aminopyrazine-2-carboxamide, to inhibit the Mycobacterium tuberculosis enzyme (MtbProRS). These derivatives show strong activity against multidrug-resistant Mtb strains and are not cytotoxic to HepG2 cells. We determined high-resolution crystal structures of MtbProRS bound to six derivatives to define their binding modes. The compounds bind in the ATP site and trigger local conformational changes that disrupt the proline pocket, supporting a dual-site mechanism of inhibition. Comparison with human cytosolic and mitochondrial ProRS reveals differences in active-site residues, electrostatics, and dynamics that explain species selectivity. Together, these structures provide a framework for designing next-generation, Mtb-selective ProRS inhibitors as new antitubercular candidates.

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Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a chronic respiratory infection that remains the leading cause of death from a single infectious agent worldwide, as reported in the WHO Global TB Report 2024. Despite the availability of a 6-month standard regimen comprising rifampicin, isoniazid, pyrazinamide, and ethambutol, the emergence and global spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb strains have severely undermined control efforts. , Current second-line agents, including bedaquiline, delamanid, and pretomanid, provide additional treatment options; however, their use is constrained by cost, toxicity, and the need for prolonged treatment. The growing prevalence of strains resistant to all available TB drugs underscores an urgent need for novel therapeutics with improved efficacy, tolerability, lower cost, and potential for treatment shortening. −

Prolyl-tRNA synthetase (ProRS), a member of the class IIa aminoacyl-tRNA synthetase (aaRS) family, catalyzes the aminoacylation of tRNAPro with l-proline, generating prolyl- tRNAPro, the substrate for ribosomal incorporation of proline during protein synthesis. ProRS is a validated drug target in multiple human diseases, including cancer, mosquito-borne infections, and diseases caused by Plasmodium and Toxoplasma parasites, among others. ProRS enzymes from several species have been structurally and functionally characterized and actively pursued in drug discovery campaigns. , Numerous ProRS inhibitors have been developed and evaluated for their potential to treat bacterial, fungal, and malarial infections, as well as for therapeutic applications in cancer and fibrosis.

Kawamoto and co-workers recently identified a series of pyrazine-scaffold inhibitors of human cytosolic ProRS (HcProRS) that target the ATP-binding site in a proline-dependent manner. Among these derivatives, the most potent, compound 1 (Figure ), binds HcProRS with a K d of 0.76 nM in the presence of l-proline. This discovery prompted the identification of additional derivatives, notably 3-benzylaminopyrazine-2-carboxamides, through a virtual screening approach. Five such derivatives (compounds 2a, 2b, 3a, 3b, and 3c) (Figure ) were confirmed to bind HcProRS by thermal shift assay (TSA), and their binding modes were determined by X-ray crystallography. Compounds bearing ortho-substituents on the benzyl ring of the C-3 amine showed measurable binding affinity toward HcProRS but exhibited limited or no significant antimycobacterial activity in vitro. In contrast, compounds bearing para-substituents on the benzyl ring of the C-3 amine did not display detectable interaction with HcProRS yet demonstrated moderate inhibitory effects on M. tuberculosis growth.

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Chemical structures of compounds targeting HcProRS and MtbProRS. Compounds 1, 2a–2b, and 3a–3c are binders of HcProRS, while compounds 4a–4g and 5a–5b target MtbProRS. Compounds shown in green represent those cocrystallized with MtbProRS in the present study.

More than 50 derivatives of 3-aminopyrazine-2-carboxamide, each containing an additional carbonyl oxygen, were subsequently synthesized and screened for antimycobacterial activity. Consistent with previous findings, series-1 compounds bearing a para-substituted benzoyl moiety on the C-3 amine and an unsubstituted carboxamide at the C-2 position of the pyrazine core showed the highest antimycobacterial potency (Table S1). Derivatives with bulky halogen (Cl or Br) or methyl substitution at the para position of the phenyl ring exhibited the strongest activity against the virulent Mtb H37Rv strain (MIC = 6.25 μg/mL for compounds 4e, 4f, and 4g). Notably, these compounds retained efficacy against MDR clinical isolates of Mtb (MIC = 12.5 μg/mL), suggesting a molecular target distinct from that of conventional antitubercular drugs. In contrast, compounds bearing meta- or ortho-substituted benzoyl groups were inactive or showed only marginal activity. Derivatives with mono- or dimethylation at the C-2 carboxamide moiety were also inactive, highlighting the importance of both hydrogens of the unsubstituted C-2 carboxamide for antimycobacterial activity. Interestingly, this contrasts with compound 1, which retains activity against HcProRS despite having only a single hydrogen at the C-2 carboxamide (Table S1).

A structural model of MtbProRS generated using AlphaFold was used for molecular docking and subsequent unbiased molecular dynamics simulations to investigate the binding of active compounds. Cytotoxicity assessment in HepG2 human hepatocellular carcinoma cells revealed that the active derivatives displayed minimal toxicity, indicating that selectivity toward mycobacterial versus human ProRS can be modulated through relatively simple structural modifications. Further optimization yielded series-2 compounds bearing an unsubstituted carboxamide at the C-2 position of the pyrazine ring, a small fluorine substituent at the ortho position (larger ortho substituents were not tolerated), and a chloro or bromo substituent at the para position of the phenyl ring. These compounds retained moderate activity against Mtb H37Rv (MIC = 12.5 μg/mL for compound 5a and 6.25 μg/mL for compound 5b) and remained active against MDR clinical isolates (MIC = 25 μg/mL for compound 5a and 12.5 μg/mL for compound 5b) (Figure ; Table S1).

In this study, we determined high-resolution crystal structures of MtbProRS in complex with the reaction intermediate prolyl-adenylate (Pro-AMP) and its nonhydrolyzable analog prolyl-sulfamoyl-adenylate (Pro-AMS), revealing key features of substrate coordination and catalytic-site architecture. To elucidate the structural basis of enzyme inhibition, we solved six additional structures of MtbProRS bound to 3-aminopyrazine-2-carboxamide derivatives from series-1 (compounds 4a–4e) and series-2 (compound 5a). These ligands occupy the ATP-binding pocket and induce active-site conformational rearrangements that disfavor proline binding. Comparative analysis with HcProRS structures highlights active-site differences, particularly in residue composition, electrostatic environment, and conformational plasticity, that underlie the observed selectivity for the mycobacterial enzyme. Together, these structural insights provide a mechanistic basis for species-specific inhibition and offer a framework for structure-guided optimization of selective MtbProRS inhibitors as potential novel antimycobacterial agents.

Results

Overall Structure of MtbProRS

We determined crystal structure of MtbProRS. The enzyme adopts a V-shaped homodimer, with monomers arranged in a head-to-tail orientation (Figure A). Each monomer comprises three domains: an N-terminal class IIa catalytic domain (residues 1–237, 416–471), a central insertion (INS) domain (residues 255–386), and a C-terminal anticodon-binding (ACB) domain (residues 488–582) (Figure A). The catalytic domain contains the three conserved motifs I, II, and III. The INS domain, characteristic of prokaryotic ProRS, is inserted between motifs II and III and forms a compact globular subdomain (Figure B).

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Molecular basis of prolyl-adenylate (Pro-AMP) recognition by MtbProRS. (A) Overall architecture of the crystal structure of MtbProRS homodimer in complex with Pro-AMP (PDB ID: 9YY6). The catalytic domain (residues 1–237 and 416–471), insertion domain (residues 255–386), and anticodon binding domain (residues 488–582) are shown in blue, yellow, and pink, respectively. Pro-AMP is depicted as sticks and the divalent calcium ions are represented as green spheres. (B) The conserved motifs I, II, and III in the catalytic domain of the MtbProRS are highlighted in blue. (C) Detailed view of the active site of MtbProRS/Pro-AMP complex. The protein backbone is shown in cartoon representation, bound Pro-AMP and the active site residues are represented as stick model. Calcium ion and ordered water molecules are shown as green and red spheres, respectively. Hydrogen bonds and calcium ion coordination bonds are shown as dashed lines.

Structural comparison shows that MtbProRS is highly similar to bacterial ProRS enzymes from P. aeruginosa (PDB ID: 8W9I; RMSD = 2.3 Å over 492 Cα atoms), Enterococcus faecalis (PDB ID: 2J3L; RMSD = 2.1 Å over 531 Cα atoms), Rhodopseudomonas palustris (PDB ID: 2I4M; RMSD = 2.6 Å over 240 Cα atoms), and Staphylococcus aureus (PDB ID: 5ZNJ; RMSD = 3.3 Å over 552 Cα atoms). , The catalytic and ACB domains are well conserved, whereas the INS domain shows conformational variability (Figure S1). Multiple sequence alignment similarly reveals strong conservation within the catalytic and ACB domains, with substantial divergence in the INS domain (Figure S2).

We determined crystal structures of MtbProRS in complex with Pro-AMP and the non-hydrolyzable analog Pro-AMS at 2.15 Å and 2.71 Å resolution, respectively. In the MtbProRS/Pro-AMP structure, the adenylate intermediate was generated in situ from l-proline and ATP present in the crystallization drop. Both complexes crystallized in the orthorhombic space group and contained two homodimers per asymmetric unit. Superposition revealed high similarity between the Pro-AMP and Pro-AMS complexes (RMSD = 0.56 Å over 589 Cα atoms; Figure S3), so subsequent descriptions focus on the higher-resolution Pro-AMP structure. Clear electron density for Pro-AMP and Pro-AMS was observed in both active sites of each structure, enabling unambiguous modeling (Figure S4). Both ligands adopt similar binding modes within the catalytic domain and are stabilized by motifs I–III (Figure B). The AMP portion adopts the canonical “bent” conformation characteristic of class II aaRSs and is coordinated by a divalent calcium ion (from calcium acetate in the crystallization conditions). The adenine ring is sandwiched between Phe157 and Arg462 (motifs II and III), with the C-6 amino group forming hydrogen bonds with Glu144 and the backbone of Ala154. Additional interactions include hydrogen bonds between N-1 and the backbone amide of Ala154, and water-mediated contacts to N-7 and N-3 (waters W53 and W18). The ribose ring adopts a C3′-endo conformation and interacts with Glu422, Gly424, and Gly459, via both main-chain and side-chain atoms of the protein.

The α-phosphate of Pro-AMP coordinates the calcium ion, which is further stabilized by His425, Glu422, and water molecules positioned by Glu211. The α-phosphate also forms an ionic interaction (salt bridge) with Arg142. The proline-binding loop adopts a closed conformation, forming a largely hydrophobic pocket (residues 200–207). The protonated α-amino group of proline in Pro-AMP forms a hydrogen bond to the side chain hydroxyl of Thr111 and an ionic interaction to side-chain carboxylate of Glu113 (class IIa TXE loop). The proline carbonyl oxygen interacts with the Arg142 side chain (Figure C). In the Pro-AMS complex, no divalent metal ion is observed near the sulfonyl group, despite magnesium formate in the crystallization solution. Instead, the sulfonyl group forms water-mediated hydrogen bonds with His425 and Glu422, and the adenine N-7 forms a direct hydrogen bond with Arg142 (Figure S5). Similar coordination has been reported for Pro-AMS–bound ProRS from E. faecalis and R. palustris (PDB IDs: 2J3L, 2I4M).

Inhibitor-Bound Structures of MtbProRS

To define how 3-aminopyrazine-2-carboxamide derivatives interact with MtbProRS, we determined crystal structures of the enzyme bound to five series-1 compounds (4a–4e) and one series-2 compound (5a), at resolutions ranging from 2.12 to 2.75 Å (Figure ; Table S2). All complexes crystallized in space group P21, with two protomers per asymmetric unit. Despite the presence of excess proline in crystallization drops, no electron density for proline was observed in any complex, except one protomer of the MtbProRS/4a structure (which has an unsubstituted phenyl ring). All six compounds occupy the ATP-binding pocket and adopt a similar binding mode. Overall, the inhibitor-bound structures are highly similar to the Pro-AMP and Pro-AMS- bound complexes (average RMSD = 1.1 Å over 575 Cα atoms), consistent with a conformational selection mechanism. Nonetheless, functionally important local rearrangements occur in the active site, as described below.

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Binding modes of series-1 and series-2 compounds in the active site of MtbProRS. Co-crystal structures of series-1 compounds(A) 4a (PDB ID: 9YYE), (B) 4b (PDB ID: 9YYF), (C) 4c (PDB ID: 9YYT), (D) 4d (PDB ID: 9YZX), and (E) 4e (PDB ID: 9Z09), and series-2 compound (F) 5a (PDB ID: 9YXS) bound to MtbProRS are shown. Compounds and interacting protein residues are depicted as stick model. Carbon atoms of the compounds and MtbProRS are colored magenta and gray, respectively. Bridging water molecules are represented as red spheres. Hydrogen bonds are indicated by black dashed lines.

PLIP analysis revealed a conserved interaction network across all inhibitor complexes. The pyrazine ring is centrally anchored by π–π stacking with Phe157 (motif II) and a cation−π interaction with Arg462 (motif III) (Figure A–F). A defining feature of all complexes is the conserved orientation of the C-2 carboxamide group: the NH2 points “up”, forming hydrogen bonds with the Glu144 side-chain carboxylate and the Ala154 backbone carbonyl (both motif II), while the carbonyl oxygen points “down” (Figure A–F). This arrangement aligns with previous SAR showing that N-methylation of the C-2 carboxamide NH2 reduces or abrogates antimycobacterial activity. This preferred carboxamide orientation appears to be intrinsic to the ligand, stabilized by an intramolecular hydrogen bond between the 3-NH2 group and the carboxamide carbonyl, and is not solely imposed by the protein. The pyrazine N-1 (N-8 in PDB files) forms a hydrogen bond with the backbone amide of Ala154, while N4 (N5) interacts with a conserved bridging water molecule, which also hydrogen bonds to the C-3 linker carbonyl and participates in an electrostatic interaction network involving Arg462 (Figure A–F).

Substitution on the phenyl ring modulates the orientation of the C-3 carbonyl. In compounds 4a, 4c, 4d, and 4e, the carbonyl remains roughly coplanar with the phenyl ring, maintaining an optimal electrostatic interaction with Arg462. In compound 4b, the bulky ortho-chloro substituent forces the carbonyl oxygen out of plane to avoid steric clash, increasing its distance from Arg462 and weakening this interaction. By contrast, compound 5a, which carries a smaller ortho-fluorine, preserves planarity and favorable contacts within the pocket (Figure F). This is consistent with prior observations that series-2 derivatives with ortho substituents larger than fluorine show reduced antimycobacterial activity. Contrary to previous predictions from molecular docking and MD simulations, the crystal structures show that the para-halogen substituents are positioned approximately 6.2 to 6.8 Å from Glu211, well beyond the distance required for halogen bonding. This observation likely reflects intrinsic structural features of MtbProRS or effects of crystal packing (Figure S6).

Superposition of inhibitor- and Pro-AMP-bound complexes shows that the pyrazine core of the inhibitors occupies the adenine-binding site, while the C-3 aminocarbonyl-linked phenyl group projects into the ribose-binding pocket (Figure ). Inhibitor binding is accompanied by a more closed active-site conformation. Met159 adopts a rotamer that moves closer to the inhibitor, and the His425 side chain shifts inward relative to its position in the Pro-AMP and Pro-AMS complexes, establishing van der Waals contacts with the phenyl ring. This is accompanied by a rotamer change in Phe427, which forms π–π stacking with His425 (Figure ).

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Structural alignment of the active site residues of the MtbProRS/compound 5a complex (gray; PDB ID: 9YXS) and the MtbProRS/Pro-AMP complex (blue; PDB ID: 9YY6). Pro-AMP, compound 5a and active site residues of MtbProRS are represented as stick model. Carbon atoms of Pro-AMP and compound 5a are colored in yellow and magenta, respectively.

In all inhibitor complexes, the proline-binding loop is disordered and could not be modeled (Figure ). No electron density for proline was observed, except for weak density in one protomer of the MtbProRS/4a complex. In that structure, proline was modeled and His425 adopts two alternate conformations, with the inward-facing rotamer at lower occupancy. Structural alignment indicates that the inward-facing conformation of His425 would sterically clash with bound proline. In addition, Arg142 (motif II), which stabilizes the proline carbonyl in the Pro-AMP complex, becomes disordered or adopts an outward orientation in the inhibitor-bound structures (Figure ). Similar outward positioning of the equivalent residue has been described in apo-ProRS structures from P. aeruginosa (PDB ID: 5UCM) and R. palustris (PDB ID: 2I4L) (Figure S7). Collectively, the inward shift of His425, displacement/disorder of Arg142, and loss of the ordered proline-binding loop generate an active-site conformation incompatible with proline binding, explaining why proline is disfavored in the inhibitor-bound state.

Structural Basis for Selective Inhibition of MtbProRS over HcProRS

Crystal structures of HcProRS bound to compound 1 (PDB ID: 5VAD) and to pyrazine-based inhibitors 2a, 2b, 3a, 3b, and 3c (PDB IDs: 7OT1, 7OT3, 7OSZ, 7OT2, 7OT0) show that these ligands bind in a proline-dependent manner and target the ATP-binding site with EC50 values (TSA) between 3.74 and 91.11 μM. , Superposition of HcProRS with MtbProRS in complex with compounds 4a–4e and 5a (average RMSD = 2.1 Å over 340 Cα atoms) reveals a globally conserved pyrazinamide binding mode. The core ring is similarly stacked between Phe1167 and Arg1278 in HcProRS (Phe157 and Arg462 in MtbProRS), and the C-2 carboxamide NH2 forms a hydrogen bond to the backbone carbonyl of Thr1164 (Ala154 in MtbProRS). N-1 of the pyrazine core forms a hydrogen bond to the backbone amide of the same residue in both enzymes (Figures A, B and S8).

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Superimposition of the (A) HcProRS/compound 1 complex (yellow; PDB ID: 5VAD), and (B) HcProRS/compound 3a complex (yellow; PDB ID: 7OT0) with the MtbProRS/compound 5a complex (PDB ID: 9YXS; gray) structures, respectively. Active site residues and binders (compounds 1, 3a and 5a) are shown as stick model. Compounds 1 and 3a are colored as yellow carbon; compound 5a is colored as magenta carbon. (C) Electrostatic surface potentials of the HcProRS/compound 3a and MtbProRS/compound 5a complexes. The region surrounding the para-substitution on the phenyl ring attached to the C3-amino moiety of the ligands is highlighted with a dotted circle.

Despite these similarities, several active-site substitutions remodel the ATP and proline-binding pocket and contribute to species selectivity. In HcProRS, Trp1169, Thr1240, Thr1276, and Leu1168 correspond to Met159, His425, Gly459, and Val158 in MtbProRS. These substitutions alter pocket shape and polarity. In HcProRS, the cyclohexyl group of compound 1 and the ortho-substituted phenyl rings of compounds 2a–2b and 3a–3c are positioned closer to the ribose-interacting loop (defined in the HcProRS/adenosine structure; PDB ID: 4K87) than in MtbProRS complexes (Figure A, B). This difference may arise from the presence of bulky Ile423 in MtbProRS versus Gly1238 in HcProRS, which restricts space on one side of the ribose pocket, while Met159 (MtbProRS) replaces Trp1169 (HcProRS) on the opposite side. These changes reorient the C-3 substituents and likely shift the position of the inhibitor carbonyl oxygen.

In HcProRS, the C-3 carbonyl group of compound 1 maintains a hydrogen bond to bridging water and forms an additional hydrogen bond with Thr1276 (Gly459 in MtbProRS) (Figure A). The polar threonine side chain can provide an extra hydrogen bond or steric constraint absent in MtbProRS. However, analogs corresponding to Mtb compound 4b, which retain this carbonyl and bind HcProRS, were found to be noncytotoxic in HepG2 cells, consistent with reduced affinity toward HcProRS. Arg1152 in HcProRS (Arg142 in MtbProRS) also behaves differently. In HcProRS, Arg1152 adopts either an inward conformation in the apo state (PDB ID: 4K86) or reorients to interact with proline (PDB ID: 7OSY) but appears unable to adopt a fully outward conformation due to steric interference from Glu1100. In contrast, in MtbProRS, Glu1100 is replaced by Ser94, allowing Arg142 greater conformational freedom, including an outward orientation that facilitates inhibitor binding in the absence of proline (Figures A, B and S8).

Thermal shift assays and docking studies have shown that compounds with para-substituted benzyl rings at the C-3 amine have potent antimycobacterial activity but fail to bind HcProRS. Our structural analysis suggests that the region of motif III that accommodates the para-substituted benzyl ring is electrostatically neutral in HcProRS but negatively charged in MtbProRS. This difference may favor interactions between para-chloro/bromo substituents and electron-rich residues in MtbProRS, potentially through halogen bonding, whereas such interactions are not supported in the neutral HcProRS pocket (Figure C). Thus, we propose that species-selective inhibition arises from a combination of residue substitutions, electrostatic differences, and distinct side-chain dynamics in the ATP and proline-binding sites.

ATP-Site Occupying Compounds Inhibit Pro-AMP Formation by MtbProRS

To assess whether compounds targeting the ATP-binding site affect MtbProRS catalytic activity, l-proline adenylation was measured in the absence of tRNAPro. MtbProRS efficiently formed the Pro-AMP intermediate under control conditions, consistent with tRNA-independent activation of l-proline (Figure A). Addition of 10 μM of each compound reduced Pro-AMP formation to varying degrees. Compounds 4c and 5a caused the strongest inhibition, with moderate to weak effects observed for the others. Residual adenylation activity of MtbProRS in the presence of 4a, 4b, 4c, 4d, 4e, and 5a was 69%, 64%, 1%, 50%, 46% and 7%, respectively (Figure B). Notably, protein aggregation might have contributed to the pronounced inhibition observed with 4c and 5a. Overall, these results indicate that compounds binding the ATP-binding site impair ATP-dependent proline activation by MtbProRS.

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(A) Pro-[α-32P]­ATP synthesis in the presence of 10 μM inhibitor and monitored by thin-layer chromatography. The rate of Pro-[α-32P]­ATP formation was determined at 37 °C over 30 min. (B) MtbProRS retained 69%, 64%, 1%, 50%, 46% and 7% adenylation activity in the presence of compounds 4a, 4b, 4c, 4d, 4e, and 5a, respectively.

Thermodynamics of Inhibitor Binding to MtbProRS

Isothermal titration calorimetry (ITC) was used to determine the thermodynamic parameters of inhibitor binding to MtbProRS. Purified protein and compounds were titrated under identical buffer conditions (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP) at 25 °C to minimize heats of dilution. Titration profiles were consistent across compounds and fit well to a one-site binding model (Figure S9; Table S3).

All six compounds bound MtbProRS with micromolar to submicromolar affinity, spanning mid micromolar (4a = 46.26 μM), low micromolar (4b = 2.80 μM, 4c = 4.53 μM), and submicromolar (4d = 0.54 μM, 4e = 0.14 μM, 5a = 0.38 μM) K d values. The K d value of compound 4a was 1–2 orders of magnitude higher compared to the other derivatives, which is consistent with the fact that 4a was the only compound that did not fully prevent the binding of Pro in the cocrystallization experiment. For all compounds, the binding was exothermic (negative ΔH) and both enthalpically and entropically favorable, indicating contributions from specific interactions and desolvation/entropy gain. Within this series, increases in antimycobacterial potency appear to track more closely with improved physicochemical properties (e.g., lipophilicity) than with large changes in intrinsic binding strength. Selected physicochemical and pharmacokinetic properties and compliance with common medicinal chemistry rules were calculated using SwissADME and MOE. The results are summarized in Table S4 in the Supporting Information. All compounds fall within drug-like chemical space and do not trigger PAINS alerts and exhibit comparable physicochemical profiles; consequently, substantial differences in permeability within this series are unlikely to fully account for the observed differences in whole-cell activity. We also estimate the probability of permeation through the mycobacterial cell wall using MycPermCheck1.1 (Table S4). All compounds showed a similar, moderate permeation probability, with only minor differences in logP resulting in comparable predicted permeation behavior. This is consistent with the high structural similarity of the series, as the used model relies on descriptors related to molecular size and shape, lipophilicity, hydrogen-bond donor count, and surface area components.

Discussion

Aminoacyl-tRNA synthetases (aaRSs) have recently gained traction as drug targets across multiple disease areas, including malaria, cancer, and bacterial and viral infections. , In TB, aaRSs are particularly attractive due to their essentiality for protein synthesis and significant divergence from human homologues, enabling selective inhibition. − The LeuRS inhibitor GSK3036656 (ganfeborole), for example, has progressed to Phase IIa clinical trials for TB. For ProRS specifically, halofuginone and related febrifugine analogs inhibit Plasmodium falciparum ProRS via an ATP-dependent, dual-site mechanism engaging both the proline and ATP binding pockets, , and Pro-AMP mimetics such as PAA-5 and PAA-38 inhibit bacterial ProRS with nanomolar potency by simultaneously occupying the amino acid and ATP sites.

More recently, bersiporocin (DWN12088) has been shown to modulate human ProRS in fibrotic tissues, selectively reducing pathological collagen synthesis while largely preserving normal protein synthesis. ,

In this study, we provide structural and mechanistic insight into inhibition of MtbProRS by 3-aminopyrazinamide derivatives that occupy the ATP pocket and sterically impact the neighboring proline-binding site. The inhibitors act as ATP-competitive, proline-independent ligands that establish a conserved interaction pattern: two hydrogen bonds between the C-2 carboxamide NH2 and Glu144/Ala154, π–π and cation−π interactions between the pyrazine core and Phe157/Arg462, and a key hydrogen bond between the C-3 carbonyl and a bridging water molecule. These interactions anchor the scaffold across multiple analogs and explain the sensitivity of activity to modifications at the C-2 carboxamide.

Inhibitor binding induces a localized conformational rearrangement in the ATP pocket, most notably the inward displacement of His425 and reorientation of Phe427. The inward-facing His425 would sterically clash with proline, and inhibitor binding is also associated with disordering or outward displacement of Arg142 and loss of an ordered proline-binding loop. Together, these changes disfavor proline binding and support a dual-site inhibition mechanism in which ATP-site engagement locks the enzyme in a conformation incompatible with substrate binding. This is mechanistically distinct from classical substrate-competitive schemes and could help limit resistance pathways that rely on simple substrate-binding-site mutations.

Our structural analysis further clarifies how ortho and para substituents on the benzyl ring modulate activity. Para-fluoro, para-bromo, and selected 2,4-disubstituted analogs containing ortho-fluoro and para-halogen groups preserve planarity of the C-3 carbonyl and maintain favorable interactions with Arg462 and the bridging water network. In contrast, bulkier ortho or meta-chloro substituents force the carbonyl out of plane, weaken electrostatic contacts, and correlate with reduced antimycobacterial activity. These findings align well with SAR from previous biochemical and microbiological studies and highlight the importance of steric compatibility in the ribose/adenine region of the ATP site.

ITC data show that binding is driven by favorable enthalpy and entropy, and that differences in biochemical affinity among this series are relatively modest compared to differences in whole-cell activity. This suggests that improved antimycobacterial potency largely reflects enhanced lipophilicity, permeability, and possibly efflux avoidance, rather than dramatic gains in intrinsic binding energy. Future optimization can therefore focus not only on strengthening key hydrogen bonds and π-interactions but also on tuning physicochemical properties for better cellular uptake and pharmacokinetics. Discrepancies between in vitro ProRS inhibition and whole-cell anti-Mtb activity may reflect several factors such as limited permeability, active efflux, and intracellular metabolism. Our MtbProRS inhibitors were derived from the human ProRS ATP-site binder, Adachi ligand, which selectively inhibits translation of proline-rich sequences in cells, providing supportive evidence that ATP-site targeting can guide effective inhibitor design.

Comparison with human cytosolic ProRS reveals that species selectivity arises from a combination of active-site residue substitutions, altered electrostatics, and side-chain dynamics. Substitutions such as Met159/Val158/Ile423 and Gly459 in MtbProRS versus Trp1169/Leu1168/Gly1238 and Thr1276 in HcProRS reshape the ATP and ribose pockets and change the orientation of C-3 substituents and their carbonyl groups. The constrained behavior of Arg1152 in HcProRS, partly due to Glu1100, versus the more flexible Arg142/Ser94 environment in MtbProRS, further explains why the same chemotype can bind MtbProRS in a proline-independent manner yet show limited interaction with HcProRS, particularly for para-substituted analogs. AlphaFold3 modeling of human mitochondrial ProRS (HmProRS) and comparison with inhibitor-bound MtbProRS revealed an RMSD of 0.86 Å over 295 Cα atoms, indicating high structural similarity. Residues in MtbProRS that interact with the inhibitors are largely conserved in HmProRS, suggesting that its ATP-binding pocket could accommodate these compounds. However, Arg182 in HmProRS (corresponding to Arg142 in MtbProRS), which participates in proline binding, could be disordered upon inhibitor binding, resulting in steric clash with the larger Glu140 in HmProRS, which is replaced by Ser94 in MtbProRS. Furthermore, the bulkier hydrophobic Tyr205 in HmProRS compared with Val158 in MtbProRS could be targeted to improve selectivity by extending compounds from the pyrazine ring side (Figure S10). Although our structures focus on MtbProRS, superposition with ProRS–inhibitor complexes from S. aureus and P. aeruginosa reveals conserved elements of binding site architecture, including positioning of key hydrogen bonds and halogen substituents relative to the adenine/ATP pocket. These parallels suggest that the design principles identified here may extend to other bacterial ProRS enzymes, albeit with species-specific adjustments. However, prior studies have shown that this scaffold series exhibits only modest antibacterial activity against E. faecalis and S. aureus, with no antifungal activity and substantially lower potency than reference standards.

Overall, the current series demonstrates promising antimycobacterial activity and mechanistic novelty. Further refinement can be guided by several structure-based strategies, including reinforcing the C-2 carboxamide hydrogen-bond network, exploiting selectivity-determining differences between MtbProRS and HcProRS and the enlarged ribose pocket, and optimizing lipophilicity and rigidity to enhance whole-cell activity. Additional strategies involve extending the scaffold to engage the adenine, ribose, and proline subsites to stabilize a nonproductive catalytic state, and introducing cyclization leading to ortho-condensed bicyclic compounds such as aminopyridopyrazines to reorganize the molecule and reinforce key intramolecular hydrogen bonds. Systematic profiling against human ProRS isoforms, combined with these structure-guided strategies, should enable the development of Mtb-selective ProRS inhibitors with improved potency, selectivity, and pharmacokinetic properties as a new class of antitubercular agents.

Materials and Methods

Expression and Purification of MtbProRS

The gene encoding MtbProRS (UniProt ID: P9WFT9) was codon-optimized for expression in Escherichia coli and cloned into the pMCSG9 vector (Twist Bioscience). The plasmid was transformed into E. coli BL21­(DE3)-Gold (Stratagene), and cells were grown in LB medium supplemented with 100 μg/mL ampicillin. Protein expression was induced at an OD600 of 0.6–0.8 with 0.35 mM IPTG, followed by incubation for 16 h at 18 °C. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM HEPES pH 8.0, 500 mM NaCl, 20 mM imidazole pH 8.0, 5% [v/v] glycerol, 10 mM β-mercaptoethanol). Following sonication and clarification by centrifugation, the supernatant was applied to Ni2+ Sepharose (GE Healthcare Life Sciences) pre-equilibrated with lysis buffer. After washing with buffer containing 50 mM imidazole, bound MtbProRS was eluted with 500 mM imidazole.

The His-tag was removed by overnight digestion with TEV protease (1:20 protease: protein molar ratio) during dialysis into 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM TCEP. The digest was passed over Ni-NTA to remove His-tagged TEV protease and residual contaminants. The flowthrough was concentrated (30 kDa MWCO) and further purified by size-exclusion chromatography on a Superdex 200 10/300 GL column (Cytiva) equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM TCEP. Peak fractions were pooled, concentrated to ∼30 mg/mL, aliquoted, and flash-cooled in liquid nitrogen.

Crystallization and Data Collection

For the Pro-AMP complex, MtbProRS (12 mg/mL) in 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM TCEP was incubated on ice for 1 h with 5 mM ATP, 5 mM l-proline, and 5 mM MgCl2. Sitting-drop vapor diffusion experiments at 16 °C were set up by mixing 0.3 μL protein–ligand solution with 0.3 μL reservoir solution (0.2 M calcium acetate hydrate, 0.1 M Tris pH 7.0, 20% [w/v] PEG 3000). Plate-like crystals appeared within 24 h and reached maximum size within 1 week.

For the Pro-AMS complex, MtbProRS (15 mg/mL) was incubated on ice for 1 h with 2 mM Pro-AMS (100 mM stock in DMSO; provided by a collaborator). Crystals were obtained at 16 °C by mixing 0.4 μL protein–ligand solution with 0.4 μL reservoir solution (0.1 M magnesium formate, 15% [w/v] PEG 3350).

For inhibitor complexes, compounds 4a–4g, 5a, and 5b were synthesized as described, , dissolved in DMSO (100 mM), and added to MtbProRS (8 mg/mL in 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) to a final concentration of 3 mM inhibitor and 3 mM l-proline. After 2 h incubation on ice, hanging-drop vapor diffusion experiments at 16 °C were set up by mixing 1 μL protein–inhibitor solution with 1 μL precipitant (0.2 M calcium acetate hydrate, 20% [w/v] PEG 3350). Crystals were cryoprotected in mother liquor supplemented with 20% (v/v) ethylene glycol and flash-frozen in liquid nitrogen. Crystallization attempts with compounds 4f, 4g, and 5b did not yield crystals of sufficient quality for X-ray analysis.

Diffraction data for all complexes were collected at synchrotron beamlines; data collection details are summarized in Table S2.

Data Processing, Structure Solution, and Refinement

Diffraction data were processed using HKL3000. The Pro-AMP and Pro-AMS complexes were solved by molecular replacement using the P. aeruginosa ProRS structure (PDB ID: 5UCM) as a search model. Inhibitor-bound structures were solved by molecular replacement using an AlphaFold2-predicted model of MtbProRS. Structures were refined in PHENIX with iterative manual model building in Coot. Ligands were fitted into Fo–Fc omit maps, and final models were validated using MolProbity. Structural analyses were performed using Coot and PyMOL (Schrödinger, LLC), and PyMOL was used for figure preparation. Data collection and refinement statistics are provided in Table S2.

Adenylation Inhibition Assay

Adenylation inhibition was assessed using a modified assay described by Bullock et al. (2003). Reaction mixtures included 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM DTT, 5% [v/v] glycerol, 1.5 μM MtbProRS, 10 μM inhibitors, and 5% [v/v] DMSO. The enzyme was first incubated with the inhibitor for 2 h at 30 °C to allow binding. The reaction was initiated by adding 100 μM ATP/0.25 μM [α-32P]­ATP, and 100 μM l-Pro, then incubating for 30 min at 37 °C. All reactions were stopped in 400 mM sodium acetate pH 5.2 with 0.1% (w/v) SDS. Aliquots were spotted onto polyethylenimine-cellulose TLC plates (Macherey-Nagel), developed in 750 mM KH2PO4 pH 3.5, and analyzed using a scintillation counter.

Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) experiments were performed using a Nano-ITC instrument (TA Instruments). MtbProRS and inhibitors were dialyzed or diluted into the same buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP). The cell contained 200 μL MtbProRS at 20 μM; the syringe contained 400 μM inhibitor. After an initial 1.75 μL injection, 19 injections of 2.35 μL were made at 300 s intervals at 25 °C.

Heats of dilution were determined from ligand-into-buffer control titrations and subtracted prior to fitting. Binding isotherms were analyzed using a one-site binding model in NanoAnalyze (version 2.4.1, TA Instruments). Thermodynamic parameters (K d, ΔH, ΔS, ΔG and stoichiometry) are summarized in Table S3.

Calculations of Basic Physicochemical Properties of the Compounds

Basic physicochemical properties were calculated using SwissADME and Molecular Operating Environment (MOE) to obtain molecular descriptors, predict pharmacokinetic parameters, and evaluate compliance with established medicinal chemistry guidelines. The probability of permeation across the mycobacterial cell envelope was estimated using MycPermCheck 1.1, based on descriptors including molecular size and shape, lipophilicity, hydrogen-bond donor count, and surface area properties. All calculated parameters are summarized in Table S4.

Supplementary Material

ao6c04844_si_001.pdf (6.2MB, pdf)

Acknowledgments

We sincerely thank the members of the SBC at Argonne National Laboratory for their help beamline setup and data collection at the SBC beamline 19-ID. We thank Dr. C. Walpole from the Structure-guided Drug Discovery Coalition (Gates Foundation) for providing Pro-AMS for these studies. Funding for the Center for Structural Biology of Infectious Diseases (CSBID) is provided by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93022C00035. The use of SBC beamlines at the Advanced Photon Source is supported by the U.S. Department of Energy (DOE) Office of Science and operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This research was also supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU. JW was supported by the Hatch program of the National Institute of Food and Agriculture, U.S. Department of Agriculture.

Glossary

Abbreviations

TB

Tuberculosis

ProRS

Prolyl-tRNA synthetase

Mtb

Mycobacterium tuberculosis

MDR

Multidrug-resistant

XDR

Extensively drug-resistant

HcProRS

Human cytosolic ProRS

TSA

Thermal shift assay

MIC

Minimum inhibitory concentration

Pro-AMP

Prolyl-adenylate

Pro-AMS

Prolyl-sulfamoyl-adenylate

PLIP

Protein–ligand interaction profiler

AaRSs

Aminoacyl-tRNA synthetases

LB

Lysogeny broth

IPTG

Isopropyl -D-1-thiogalactopyranoside

HEPES

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

TCEP

Tris­(2-carboxyethyl) phosphine hydrochloride

TEV

Tobacco etch virus

ITC

Isothermal titration calorimetry

DMSO

Dimethyl sulfoxide

SDS

Sodium dodecyl sulfate;

The crystal structures of MtbProRS bound to Pro-AMP, Pro-AMS, 4a, 4b, 4c, 4d, 4e, and 5a have been deposited in the PDB under the accession codes 9YY6, 9YYO, 9YYE, 9YYF, 9YYT, 9YZX, 9Z09, and 9YXS, respectively. The authors will release the atomic coordinates upon the article’s publication.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c04844.

  • Supporting Information includes tables with summary of antimycobacterial activity of published compounds; X-ray data collection and structure refinement statistics; thermodynamic parameters from ITC analysis; estimated mycobacterial cell wall permeability for discussed compounds; figures with structural comparison of the MtbProRS with homologous enzymes, multiple sequence alignment of ProRS from selected bacteria; superimposition of the monomeric structures of MtbProRS bound to Pro-AMP and Pro-AMS; electron density maps for discussed ligands; detailed view of the active site of the MtbProRS/Pro-AMS and MtbProRS/compound 4d complexes; structural comparison MtbProRS/Pro-AMP complex with apo-structures of ProRS from P. aeruginosa and R. palustris; comparison structures of complexes HcProRS/compound 2a and the MtbProRS/compound 5a; ITC thermograms showing the ligand binding interactions; and structural comparisons of the active site residues of MtbProRS/compound 4d and AF3 generated model of HmProRS (PDF)

#.

Eli Lilly and Company, Indianapolis, IN 46285, USA

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao6c04844_si_001.pdf (6.2MB, pdf)

Data Availability Statement

The crystal structures of MtbProRS bound to Pro-AMP, Pro-AMS, 4a, 4b, 4c, 4d, 4e, and 5a have been deposited in the PDB under the accession codes 9YY6, 9YYO, 9YYE, 9YYF, 9YYT, 9YZX, 9Z09, and 9YXS, respectively. The authors will release the atomic coordinates upon the article’s publication.

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