Discovery of potent antimycobacterial agents targeting lumazine synthase (RibH) of Mycobacterium tuberculosis

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.

Tuberculosis (TB) remains a leading cause of death globally 1,2 .The current standard TB treatment regimen involves a combination of multiple drugs taken over a long duration of 6 to 9 months 1,2 .The lengthy treatment duration escalates the risk of patient non-compliance, leading to treatment failure and the development of drug resistance 1 .The mounting challenges with current anti-TB therapies have hindered the efforts to eliminate TB by 2030 and have created an urgent demand for the development of novel and impactful antibiotics 2 .Mycobacterium tuberculosis (M.tb) is highly vulnerable to developing resistance mutations against existing drugs 2,3 , as the majority of these drugs have one or few target(s) which allow pathogens to evolve mechanisms for evading or offsetting the inhibitory effects 3,4 .Hence, the development of drugs targeting new pathways is considered one of the important strategies to circumvent the current drug resistance mechanisms 5,6 .Further these new drugs may have the potential to yield synergism with existing first-line anti-TB drugs leading to enhanced treatment outcomes, allowing shorter, more efficacious regimens with lesser side-effects 7 .
We next docked our in-house library consisting of ~ 3000 compounds and ~ 0.57 million compounds from the ASINEX screening library (Asinex Corp.), to identify the leads with the highest docking scores and desirable absorption, distribution, metabolism, and excretion (ADME) properties as described in methods.On molecular docking, we identify fifty-five compounds (40 from our in-house library and 15 from the Asinex library) (as listed in Supplementary Table 1) with the best binding orientations (see methods for details), docking scores, and ADME values.In silico analysis of the lead compounds targeting RibH predicts these compounds to possess good ADME properties (Table 1), bioavailability, gastrointestinal absorption and are also predicted to have a better cellular retention as they do not induce P-glycoprotein efflux pumps, unlike rifampicin [30][31][32] .Hence, RibH targeting drugs are predicted to have good pharmacokinetic properties with minimum drug-drug interactions and toxicity (Table 1).
The lead compounds selected by us in this study have not been reported earlier and hence, were selected for further assessment of antimycobacterial activity.

Antimycobacterial activity of the short-listed compounds
After the preliminary high-throughput virtual screening, fifty-five short-listed compounds were screened against M. tb H37Rv strain to evaluate their minimum inhibitory concentration (MIC) using microplate Alamar Blue assay (MABA) (Fig. 3A-C).Among the 9 compounds showing MIC in the range of 0.78-12.5 µg/ml in the primary screen, A52 is found to be the most potent with an MIC of 0.78 µg/ml (Fig. 3 C, D, Supplementary Table 1), whereas the MIC of both A10 and A39 is 1.56 µg/ml.Out of the 55, 12 compounds show moderate anti-tubercular activity in the range of 25-50 µg/ml and thirty-four show poor activity with MIC > 50 µg/ml, despite the high docking score (Supplementary Table 1).

Characterization of the top hits exhibiting anti-mycobacterial activity
As shown in Supplementary Table 1, we identified three compounds, namely A10 ( 5 exhibit the best minimum inhibitory concentration (MIC) among all the 55 compounds tested against M. tb strain H37Rv in the range of 0.78-1.56µg/ml (Supplementary Table 1).Strikingly, these compounds appear to be similar in their basic structure (see supplementary methods), with minor modifications in the R group resulting in different binding attributes (for synthesis scheme, see supplementary methods).The compounds namely, A10, A39, and A52, show a docking score of −7.276, −6.825, and −8.431 kcal/mol, respectively, signifying that these molecules can tightly bind to the RibH active site (Supplementary Table 1).
As shown in Fig. 4 riboflavin binding pocket analysis reveals that, riboflavin forms several hydrogen bonds with amino acid residues His 28, Ala 59, Glu 61, Val 81, Ile 83 of chain A, and Asn 114 and Lys 138 of chain B (Fig. 4A).Binding pocket analysis for the short-listed compounds, namely, A10, A39 and A52 reveals multiple interactions of these compounds with the active site amino acids (Supplementary Table 1).Interaction of A10 with the binding groove shows formation of two hydrogen (H) bonds (Fig. 4B).The amide-carbonyl of A10 interacts with Ala 59 and the amide-NH interacts with Val 81 forming H-bonds of bond length 2.28 Å and 2.33 Å,  , all the compounds are predicted to have moderate to good solubility with high gastrointestinal (GI) absorption.The Lipophilicity is > 1 and < 5, indicating good oral absorption.Bioavailability of 0.55 indicates these drugs to be good candidates for further preclinical evaluation.None of the drug molecules are substrate for P-glycoprotein induction hence, are going to be bioavailable and have good bio distribution.As only 3 out of 5 CYP450 family of enzymes are inhibited suggesting that drugs may have high intracellular retention, but can be metabolized by the residual 2 CYP450 enzymes and can possibly be cleared by the renal system.
They are anticipated to have low drug-drug interaction and low toxicity.None of the compounds are predicted to cross BBB-blood brain barrier.Microscale thermophoresis (MST) reveal a high binding affinity of the shortlisted compounds with purified lumazine synthase.
We next assessed the binding affinity of the three shortlisted compounds with the purified lumazine synthase by determining the dissociation constant (K d ) with the help of MST assay in vitro.Details of cloning, purification, and characterization of RibH protein utilized in this assay are described in the Supplementary methods.In the assay conditions described, MST analysis reveal that riboflavin, A10, A39, and A52 bind to lumazine synthase with a binding affinity (K d ) of 86 nM, 69 nM, 2.3 nM, and 48 nM, respectively (Fig. 5A-H).

In vitro riboflavin displacement assay reveals specific binding of the short-listed compounds to RibH active site
We next verified if the short-listed compounds bind specifically to the active site of RibH as predicted by our in-silico analysis.For this, a simple and sensitive assay developed by Chen et al. 29 is utilized.The assay is based on the displacement of the bound riboflavin (non-fluorescent) from the active site by a compound competing for the same binding site resulting in release of the free riboflavin into the solution that fluoresces at Ex 440nm/ Em 530nm .First, 27.04 µM of riboflavin is pre-complexed with 28 µM of RibH protein as described in Supplementary methods (~ 0.966 times molar ratio of riboflavin w.r.t the protein concentration).The complex is then diluted to a final concentration of 4 µM and the short-listed compounds are added at a concentration of 12.5 µg/ml (Supplementary methods) followed by fluorescence measurement (n = 8).Under the given test conditions, the three compounds, namely, A10, A39 and A52 exhibit ~ 17.5%, ~ 25.5% and ~ 19.98% displacement of riboflavin from the RibH protein, respectively (Supplementary Fig. 2) indicating the ability of these compounds to specifically compete and displace riboflavin bound to M. tb RibH protein from the active site.Moreover, displacement caused by these compounds corroborates the MST observations.

Reduced FAD + levels in M. tb treated with RibH binding ligands
To further establish the specific action of RibH binding compounds as inhibitors of the M. tb riboflavin biosynthesis, intracellular FAD + levels are measured for the untreated and the treated H37Rv cultures by high-performance liquid chromatography (HPLC).Following treatment of M. tb H37Rv with the short-listed compounds, extracted nucleotides are subjected to HPLC analysis, wherein, FAD + peaks are monitored on photodiode array detector and identified by their retention times (~ 1.9 min) at a wavelength of 450 ± 2 nm with reference to a solution of FAD + as standard (Supplementary Fig. 3A-E).Treatment of M. tb with shortlisted compounds resulted in reduced intracellular levels of FAD + compared to the untreated M. tb control group (Supplementary Fig. 3F, G).Particularly, treatment with A10, A39 and A52 resulted in markedly reduced absorbance (mAU) values and area under curve (AUC) represented as area ratio (with respect to internal control cXMP) compared to the untreated M. tb (Supplementary Fig. 3E).Among the three compounds, A52 exhibit the maximal reduction in FAD + levels compared to the other two compounds, which corroborates well with its highest in vitro anti-mycobacterial activity (Fig. 3C).Hence, reduced levels of FAD + on treatment with RibH binding compounds, MST derived binding affinity and riboflavin displacement assay support our hypothesis that compounds discovered in this study, act through specific binding and inhibition of lumazine synthase (RibH) and riboflavin biosynthesis of M. tb leading to reduced production of the final product FAD + , which leads to the anti-mycobacterial activity of these compounds.

Drugging lumazine synthase results in synergistic action with the first-line anti-TB drugs
We sought to determine if: (i) the anti-mycobacterial action of A10, A39, and A52 can be enhanced by combining with the first-line anti-TB drugs, and (ii) the addition of these compounds at the sub-MIC level increases M. tb susceptibility to the first-line anti-TB drugs.For this, a checkerboard MABA 33 was employed, wherein A10, A39, and A52 were tested at various concentrations above and below MIC in combination with different concentrations at or below MIC of rifampicin (0.19-0.006 µg/ml) (Fig. 6A-C) and isoniazid (0.39-0.012 µg/ml) (Supplementary Fig. 4A-C) against M. tb H37Rv.Strikingly, the addition of all three RibH-targeting drugs show synergistic action with rifampicin dramatically improving its MIC to ≤ 0.006 µg/ml, with 15-, 8-, and 7.5-fold reduction by A10, A39, and A52, respectively.The addition of rifampicin also enhances the antimycobacterial activity of A10, A39, and A52 resulting in a reduction in MIC by 4-, 4-, and 2-fold, respectively.Similarly, A10, A39, and A52 improve the efficacy of isoniazid with 15.8-, 32.5-and 15.8-fold reduction in MIC, respectively.Table 2 depicts the fractional inhibitory concentration index (FICI), wherein combining these compounds with rifampicin and isoniazid results in synergistic action with FICI ≤ 0.5.

Intracellular growth reduction of M. tb by RibH inhibitors
To understand if these potent molecules can traverse the barrier of the host cell membrane to effectively reduce the intracellular growth of mycobacteria inside the macrophages, the antimycobacterial activity of A10, A39, and A52 is examined at various concentrations above and below the MICs (3.125, 1.56, 0.78 and 0.39 µg/ml), as described in methods.Infection of human macrophage line THP-1 by M. tb H37Rv and drug treatment is performed for 5 days, and on the 6 th day infected macrophages are lysed and colony forming units (CFU) are determined.Our results show that all the compounds are highly effective in reducing the growth of M. tb, as evident from significant decrease in colony forming units (CFU) in the case of A10, A39, and A52, respectively, compared to the untreated infected cells (p < 0.0001) (Fig. 7, Supplementary Table 6) Importantly, A52 is found to be the most potent among all the compounds that we tested across various concentrations causing 1.2-1.9log 10 reduction in CFU as compared to A39 (1.09-1.2log 10 reduction in CFU) and A10 (0.73-1.2 log 10 reduction in CFU).

Lumazine synthase targeting drugs interfere with resuscitation in an in vitro dormancy model
We next tested, if RibH targeting drugs function against nutritionally starved M. tb in an in vitro dormancy/ non-replicating persistence model (Supplementary Fig. 5), and whether these drugs interfere with the ability of the bacteria to resuscitate when exposed to the nutrient-rich conditions.We mimicked dormancy by starving M. tb H37Rv in phosphate-buffered saline for a period of six week, and then exposed the bacteria to A10, A39, and A52 with rifampicin and isoniazid as control drugs.We followed M. tb growth by most probable number (MPN) assay, as described in the methods.Remarkably, our findings reveal that these drugs not only act upon the nutritionally starved bacteria but also interfere with their ability to resuscitate despite the presence of nutrient-rich conditions, which otherwise favours bacterial growth in the absence of drugs.All the compounds show potent activity against nutritionally starved M. tb H37Rv (Fig. 8), however, A10 causes maximum growth inhibition resulting in ~ 2.9 log 10 reduction in CFU compared to that of untreated control (p < 0.0001).Noteworthy to mention, A10 performs better than the first-line anti-TB drugs, resulting in 0.6 log 10 and 1.081 log 10 fewer bacillary counts compared to rifampicin and isoniazid (p < 0.01), respectively (Fig. 8).A10 also shows better activity Figure 6.Anti-mycobacterial activity of short-listed compounds in combination with first-line anti-TB drug rifampicin.The figure depicts the representative pictograms obtained on screening of anti-mycobacterial activity of three of the shortlisted compounds against M. tb H37Rv in combination with rifampicin by MABA.The assay was performed at least twice with two technical replicates in each case.In each case (A-C) rifampicin was tested at various concentrations between 0.19 and 0.006 µg/ml (two-fold serial dilution) either alone or in combination with various concentrations of (A) A10, (B) A39 and (C) A52.These shortlisted compounds were either tested alone at various concentrations between 3.125 and 0.09 µg/ml (two-fold serial dilution) or at their respective MICs and sub-MIC concentrations in combination with various concentrations of rifampicin (MIC to sub-MIC range).A synergistic enhancement in antimycobacterial activity of rifampicin and shortlisted compounds was observed as described in Table 2 (for combined anti-TB activity with Isoniazid see supplementary Fig. 4).
Table 2. Synergistic interactions between RibH targeted drugs and first line anti-TB drugs rifampicin and isoniazid.Table describes degree of interaction of pairwise combinations of rifampicin and isoniazid in combination with A10, A39 and A52 against M. tb H37Rv.Fractional inhibitory concentration index (FICI) was calculated using formula = FIC A + FIC B. FIC A = MIC of compound A in combination with B/MIC of compound A alone.FIC B = MIC of compound B in combination with A/MIC of compound B alone.Synergy is defined as FICI of ≤ 0.5, antagonism as ≥ 4. #, Stand-alone MIC of the compound; *, MIC of the compound in combination; Fractional inhibitory concentration (FIC) for each compound is calculated considering the observed MICs of rifampicin/isoniazid and the short-listed compounds in each specific experiment.

RibH-a highly conserved drug target across various mycobacterial species
Having established the essentiality, druggability, synergism with existing drugs and safety profile of drugs targeting RibH of M. tb we next analysed if RibH is conserved during evolution among various mycobacterial species belonging to Mycobacterium tuberculosis complex (MTC) and Non-tuberculous mycobacteria (NTM).We first conducted multiple sequence alignment of RibH protein for various mycobacterial species and determined the phylogenetic relationship between them (Supplementary Figs. 6 and 7).RibH protein was found to be highly conserved in nature across the MTC, NTM, and Mycobacterium leprae (M.leprae) with minimum divergence.We then specifically compared the crucial amino acids present in the drug binding and co-crystal ligand binding pocket across various mycobacterial species.Remarkably, the binding site for A10, A39, A52, and TP6 was found to be highly conserved (95-100% identity) across the mycobacterial species (Supplementary Table 2).Especially, major mycobacterial pathogens were found to have 100% identity in the drug-binding pocket, thereby increasing the likelihood of broad-spectrum activity of these drugs for treating various other mycobacterial infections.Interestingly, drug binding pocket was also found to be conserved in M. leprae (causative agent of leprosy in humans and belonging to Mycobacterium leprae complex MLC).The conserved nature of the RibH protein and drug binding pocket increases the translational potential of the drugs developed by us, making it suitable for future testing against MTB, NTMs, and MLC with broad spectrum applications for treating human and animal diseases caused by mycobacteria.

Discussion
This study describes a comprehensive investigation into the role of lumazine synthase (encoded by ribH) in mycobacterial growth and the potential for therapeutic targeting of M. tb RibH towards developing new anti-TB agents.In this study we also utilize CRISPRi to validate the drug target by conditionally silencing the ribH gene in M. tb leading to significantly reduced ribH expression and growth defects.The growth defects observed in the ribH knockdown strain were rescued by addition of an excess amount of exogenous riboflavin (although, much above the physiological concentration).These observations highlight the key role of riboflavin biosynthesis for mycobacterial survival and confirm specific nature of CRISPRi based interference of this pathway via lumazine synthase.
We then identified drugs targeting the riboflavin biosynthesis pathway, a crucial source of important cofactors-FAD and FMN in M. tb.Among the various enzymes involved in riboflavin biosynthesis, we identified RibH to be the most suitable candidate for several reasons.First, it was found to be highly conserved across the evolutionary scale of mycobacteria (Supplementary Figs. 6 and 7 and Supplementary Table 2).With only a single copy of this gene in the M. tb genome and no evidence for redundancy in its enzymatic function, interference at H37Rv cultures were starved for 6 weeks in phosphate buffered saline, followed by treatment with the compounds namely, A10, A39 and A52 or first-line anti-TB drugs isoniazid or rifampicin at an equimolar concentration of 10 µM.Bacterial count estimation was carried out using the MPN (most probable number) assay.CFU data (n = 3) is represented as geometric mean ± SEM log 10 CFU/ml.Data represents geometric mean with 95% CI for (n = 3).All the three compounds gave significant growth inhibition as compared to untreated control (p < 0.0001, two-tailed t-test using GraphPad Prism software).Compound A10 shows significant reduction in M. tb growth as compared to the first line anti-TB drug Isoniazid (p < 0.05, oneway ANOVA using GraphPad Prism software).www.nature.com/scientificreports/ the penultimate step of riboflavin synthesis catalysed by lumazine synthase is anticipated to substantially curtail the pathway.The reduced levels of FAD + observed in our study on treating M. tb with the short-listed RibH inhibitors, antimycobacterial action of these drugs and reduced growth of ribH-KD further substantiate our hypothesis.The latter, also corroborates well with the ribH gene essentiality reported in the TraSH screens 9,12 .
Considering the crucial role of cofactors derived from this pathway in diverse metabolic functions, interference of this enzyme employing specific drug molecules possibly makes M. tb metabolically unfit leading to cell death.In this study, our high-throughput molecular docking approach allowed us to screen ~ 0.6 million compounds capable of binding to RibH.Through this screening process, we discovered three highly potent compounds that exhibit promising antimycobacterial activity against the M. tb H37Rv as standalone drug.In absence of commercially available substrates of lumazine synthase, while direct enzymatic inhibition assays could not be performed, we employ alternative step-by-step approach, to provide conclusive evidence for (i) binding of these short-listed compounds to RibH with high affinity using MST, (ii) specific binding of these compounds to the active site by competitively displacing riboflavin, its natural ligand by riboflavin displacement assay and (iii) metabolic inhibition of riboflavin biosynthetic pathway causing diminished production of FAD + , the end product of the pathway employing HPLC based relative estimation.
The most noteworthy finding of our research was the potential synergism of these compounds with the first-line anti-TB drugs, rifampicin, and isoniazid in vitro.Although, the detailed mechanism of action of drugs are currently not known to us, future studies to understand the changes in mycobacterial cell wall, and other transcriptional and metabolic profiles will provide mechanistic insights into the increased vulnerability of M. tb to the first-line anti-TB drugs on exposure to RibH targeting drugs.
The first-line anti-TB drugs are known for their high toxicity and side effects in 30-40% of the patients, resulting in non-compliance to TB therapy 34,35 .In silico analysis of lead compounds targeting RibH predicts these compounds to possess good ADME properties (Table 1), bioavailability, and gastrointestinal absorption and are also predicted to have better cellular retention as they do not induce P-glycoprotein efflux pumps, unlike rifampicin [30][31][32] .Hence, RibH-targeting drugs are predicted to have good pharmacokinetic properties with minimum drug-drug interactions and toxicity (Table 1 and Table 3).Possibly a better cellular retention due to non-induction of P-glycoprotein 36 would have contributed to an effective intracellular mycobacterial clearance as observed in this study (Fig. 7 and Table 3).Our study also reveals that when drugs were tested in the presence of riboflavin at physiological concentrations, there was no significant change in the respective MICs (Supplementary Fig. 8), indicating the limited ability of M. tb to restore growth post-drug treatment.Additionally, exogenous supplementation with 10 µM riboflavin or the cocktail of FMN and FAD (2.5 µM each) failed to rescue the growth defects observed in the knockdown strain (Supplementary Fig. 9A).However, it is noteworthy that only supplementation with an extraordinarily excessive amount of riboflavin (20 µM, well beyond physiological concentrations) successfully rescued the growth of the ribH-KD strain (Supplementary Fig. 9B).These findings highlight the intricate relationship between riboflavin supplementation and M. tb growth and underscore the importance of targeting riboflavin biosynthesis for anti-TB drug development.
Most of the anti-TB drugs such as rifampicin and isoniazid used to treat TB caused by M. tb, are primarily effective against actively replicating bacteria.However, their activity declines against non-replicating persister cells when tested in an in vitro dormancy model 37 .Since, earlier studies reported increased expression of RibH in oxidative stress and non-replicating persistent M. tb 38 , we exposed nutrient-starved non-replicating M. tb to RibH targeting drugs and provide evidence for interference with M. tb resuscitation.
Having established, M. tb RibH being a potential drug target, our study is anticipated to promote future research toward designing compounds with better fit, activity, and ADME properties.Given the limitations of rifampicin and isoniazid against non-replicating persister cells, and improved MIC of rifampicin by combination therapy observed in our checkerboard MABAs, combining multiple drugs with different mechanisms of action, could achieve effective clearance of both actively replicating bacteria as well as non-replicating persister cells, thereby improving the treatment outcomes.Future studies comparing and combining these newly discovered drugs with other anti-TB drugs, such as pyrazinamide, fluoroquinolones 39 (e.g., moxifloxacin), or bedaquiline 40 which have demonstrated better activity against NRP would reveal their synergy and interactions with drugs of the anti-TB regimen, other than rifampicin and isoniazid.As RibH protein and the drug binding sites are found to be highly conserved, the leads identified in this study have the potential to tackle a variety of geographically widespread M. tb lineages causing active, persistent, and reactivation TB 3 .The combination of these compounds with existing first-line anti-TB drugs also presents a potential strategy for overcoming drug resistance and enhancing treatment outcomes.Future preclinical studies are warranted to evaluate the full potential of these compounds and study their impact on TB control.

Ethics statement
All the in vitro studies involving risk groups 3 and 2 M. tb are done as per the guidelines and approval of the institutional biosafety committee.

Construction of knockdown strains
To achieve the repression of ribH, a pair of complementary oligonucleotides (Cr_UP: GAT CTT TCC GTG CCA GCT G and Cr_DN: CGC AGC TGG CAC GGA AAG ATC CAT G) specific to Rv1416 ORF between 73 and 93 bp downstream to the 5' end, were synthesized, annealed and cloned in a hygromycin-resistant (listed in Supplementary Table 4), E. coli-mycobacteria shuttle plasmid pGrna2 at SphI-AclI sites, as previously described 42,43 , and illustrated in Fig. 1.The recombinant pGrna2 plasmid containing Rv1416-specific guide sequence was transformed into a kanamycin-resistant M. tb mc 2 7902::pTetint-dcas9 43 to generate knockdown strain, namely, ribH-KD.Suppression was achieved by treatment of bacterial cultures with various concentrations of Anhydrotetracycline (ATc) in the range of 50-100 ng/ml for 4-9 days (unless indicated otherwise).

In vitro growth analysis
Growth of ribH-KD strain of M. tb was monitored in 7H9 broth in the absence or the presence of 50 ng/ml and 100 ng/ml anhydrotetracycline (ATc) at 37 °C, 200 rpm.The growth was monitored by measuring optical density spectrophotometrically at a wavelength of 600 nm (OD 600nm ) for 9 days.The optical density was used to create time-stamped growth curves.

RNA extraction and quantitative RT-PCR
The total RNA was extracted from the M. tb using Trizol reagent (Sigma) and purified using RNeasy Plus mini kit (Qiagen) as per the instructions given by the manufacturer.cDNA synthesis was done using the iScript cDNA synthesis kit (Bio-Rad).Then the qRT-PCR was performed with iTAq Universal SYBR Green Supermix (Bio-Rad), employing ribH gene-specific primers, and 15 ng cDNA.Gene expression was quantified using the qRT-PCR system (Roche LightCycler 480).The expression of ribH in the knockdown strain was compared to the parent strain (mc 2 7902) and sigA was used as an internal control for normalization.The primers used are listed in Supplementary Table 4.

Computational details
In silico analyses were performed in Dell Precision T7610 workstation (8 processors; 8 GB RAM; ZOTAC 3 GB graphics; Maestro 9.8, Schrodinger, New York, U.S.A) workstation running on Redhat 6.1 Linux environment.Molecular docking calculations were conducted using two different strategies i.e., pharmacophore-based virtual screening (PBVS) and Structure-based flexible docking using Glide application in the Maestro 9.8 software package (Schrodinger, LLC, New York, 2015).The BITS in-house and Asinex compound libraries were first docked against M. tb RibH using PBVS approach by e-pharmacophore generation and validation leading to the identification of 15 molecules from Asinex library and 14 from BITS in-house library.We also performed blind docking on the BITS in-house library and short-listed an additional 26 molecules.

Protein preparation
Crystal structure of M. tb lumazine synthase (encoded by ribH, Rv1416) with Protein Databank (PDB) ID 2C92 bound to 3-(1,3,7-trihydro-9-d-ribityl-2,6,8-purinetrione-7-yl) pentane 1 phosphate (TP6) having a resolution of 1.60 Å was considered for this study.There are other crystal structures published for this protein (with PDB IDs-1W19, 1W29, 2C94, 2C97, 2C9B, 2C9D, and 2VI5), however we have used 2C92 due to the best crystal structure resolution among all the published structures 19 .The protein exists in homopentameric form with substrate binding sites at the interface of two protein chains/subunits, hence for protein preparation and all the calculations, the complete dimer was used.Protein was prepared using the protein preparation wizard of Maestro 9.8, Schrodinger, New York, U.S.A.The co-crystal structure was pre-processed and water molecules within 5 Å distance from the ligand were removed, missing hydrogens and loops were added using Schrödinger protein preparation wizard.In addition, protein preparation also involved the addition of the bond orders and formal charges along with hydrogens to the hetero groups of the protein.Followed by an energy minimization to a convergence of RMSD 0.30 Å using OPLS_2003 as a force field.

Compound libraries
For identifying lead compounds that could potentially inhibit the enzymatic activity of lumazine synthase and hence serve as an anti-mycobacterial agent, we utilized two libraries, the first library consists of ~ 3000 compounds from our BITS (Birla Institute of Technology and Science) in-house database containing compounds that have ADME properties commonly observed in drug and drug-like compounds 36 and ASINEX compound library comprising of ~ 0.57 million compounds (https:// www.asinex.com/ scree ning-libra ries-(all-libra ries)).www.nature.com/scientificreports/Hypothesis generation for energy-optimized structure-based e-pharmacophore Glide energy grids were generated for the prepared protein-ligand complex.The binding site of RibH was defined by a rectangular box surrounding the ligand in the X-ray structure.The ligand was refined using Glide and default settings were used for the refinement and scoring.Starting with the refined ligand, pharmacophore sites were automatically generated with Phase with the default set of six chemical features: hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic (H), negative ionizable (N), positive ionizable (P), and aromatic ring (R).Hydrogen bond acceptor sites were represented as vectors along the hydrogen bond axis in accordance with the hybridization of the acceptor atom.Hydrogen bond donors were represented as projected points, located at the corresponding hydrogen bond acceptor positions in the binding site.Projected points allow the possibility for structurally dissimilar active compounds to form hydrogen bonds in the same location, regardless of their point of origin and directionality.Each pharmacophore feature site is first assigned an energetic value equal to the sum of the Glide XP contributions of the atoms comprising the site.This allows sites to be quantified and ranked based on these energetic terms.

Database preparation
The commercial chemical ASINEX virtual library with over 600,000 compounds & in-house 3000 compounds library were processed to avoid redundancy and Lipinski filters were applied to select compounds that have drug-like properties.The purpose of the redundancy check is to avoid structures with the same SMILES notation (Singleline entry system for structures) and also to remove conformers of very similar conformation using an RMSD cutoff of 1.0 Å. Database molecules were prepared using LigPrep and Epik to expand protonation and tautomeric states at pH 7.0 (± 2.0 pH units).For each ligand, LigPrep generates a maximum of eight tautomeric forms.If for a compound, chirality is already specified, it is retained.However, if the compound chirality is undefined, at most 32 stereoisomers were generated for each ligand used for screening.Conformational sampling was performed for all database molecules using the ConfGen search algorithm.We employed ConfGen with the OPLS_2005 force field and a duplicate pose elimination criterion of 1.0 Å RMSD to remove redundant conformers.A distance-dependent dielectric solvation treatment was used to screen electrostatic interactions.
A maximum relative energy difference of 10.0 kcal/mol was chosen to exclude high-energy structures.Using Phase, the database was indexed with the automatic creation of pharmacophore sites for each conformer to allow rapid database alignments and screening.

E-pharmacophore database screening
To dock the compound libraries into the generated grid, the libraries were first pre-processed to create an e-pharmacophore.E-pharmacophore is defined as a hypothetical ideal 3D orientation or spatial and electronic characteristics of the functional groups of a molecule necessary to ensure optimal interactions with a specific target protein to achieve a biological response.For the e-pharmacophore approach, explicit matching was required for the most energetically favorable site, with a scoring cut-off better than −1.0 kcal/mol.Screening molecules were required to match a minimum of four sites.Distance matching tolerance was set to 2.0 Å as a balance between stringent and loose-fitting matching alignment.Database hits were ranked in the order of their Fitness score as implemented in the default database screening in Phase.We validated the pharmacophore by docking the co-crystal ligand TP6.E-pharmacophore generation involves two steps, in the first step based on the co-crystal structure of the protein with a ligand (TP6), e-pharmacophore features such as hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic (H), negative ionizable (N), positive ionizable (P) and aromatic ring (R) were assigned to the bound ligand using the Glide tool.

Molecular docking
Database ligands were docked into the binding sites of RibH with Glide utilizing the high-throughput virtual screening (HTVS) scoring function to estimate protein-ligand binding affinities.The centre of the Glide grid was defined by the position of the co-crystallized ligand.Default settings were used for both grid generation and docking.Compounds with best docking and GLIDE scores were then subjected to GLIDE XP screening.Docked protein and ligand datasets were visualized and molecular surface complex pictures were generated using Maestro.Using this tool, we identified 17 compounds from ASINEX.

Grid generation
The receptor site or the active site at the interface of two subunits, where the inhibitors are to be docked was generated using the 'generate grid' sub-application of the Glide tool in Maestro 9.8.For the generation of the receptor grid, active site residues were selected to locate the coordinates of the receptor center.The grid has default parameters of van der Waals scaling factor of 1.0 and charge cut off of 0.25 subject to an OPLS_2003 force field.The generated grid was then utilized as an active site for docking [in-house database of 3000 compounds] using the 'extra precision' (XP) flexible docking method located in the Glide tool.Using this tool, we further identified 26 compounds.For evaluating and ranking the shortlisted compounds, we utilized two-step method.First, a docking score cut-off of −6 was applied and any molecule from the libraries with a docking score > −6 (Between 0 to −6) were excluded.Next, the shortlisted compounds were further evaluated on the basis of their molecular interactions with the important amino acids (29 amino acids) in the binding site of RibH.These amino acids were selected based on the co-crystal ligand interactions.Hence, a docking score < −6 plus and interaction with at least 3 amino acids were further shortlisted assuming a higher probability of binding to the substrate binding site.
Vol:.( 1234567890 MIC for the new compounds identified through in silico analysis was carried out using methods as described 44 .Briefly, M. tb strain H37Rv (ATCC27294 strain) was cultured in Middlebrooks 7H9 broth (Himedia) supplemented with 10% OADC (Himedia) and 0.05% Tween-80 (Sigma) for 4-5 days.The culture was grown to the mid-log phase until an OD 600nm of 0.4-0.8.The culture was then diluted to obtain an OD 600nm of 0.01 corresponding to ~ 10 6 CFU/ml.Based on the docking score and chemical structure 40 compounds were selected from BITS in-house library and 15 compounds from the ASINEX compound library (Supplementary Table 1).These compounds along with standard anti-TB drugs-rifampicin (Himedia) and isoniazid (Sigma) were dissolved in absolute DMSO at a concentration of 2 mg/ml to prepare the drug stock.The drug stock was then serially diluted in a 96-well plate to achieve a final concentration in the range of 50 µg/ml to 0.006 µg/ml.To each well containing the desired concentration of compounds or standard anti-TB drugs, 0.1 ml of the bacterial culture (~ 10 5 CFU/well) was added in a final volume of 200 µl.The plate was then sealed and incubated at 37 °C for 6 days.For the Alamar Blue assay, a mixture of resazurin sodium salt (Sigma) and Tween-80 was freshly prepared in autoclaved single distilled water to obtain a final concentration of 1.27 mM and 8%, respectively and the solution was then filter sterilized using 0.2 µ syringe filter.On day 6, 30 µl of this freshly prepared sterile solution is added to each well of the plate and incubated for 16-18 h at 37 °C in the dark.Color change from purple to pink is noted and photographed, wherein, purple color indicates a lack of growth and pink indicates live bacteria.MIC is determined from the minimal concentration of the antibiotics or compounds exhibiting purple color.MIC shown in Supplementary Table 1 solely reflects the data w.r.t.primary screening of the compounds in a single run.Based on this primary round, compounds with MIC ranging between 6.25 and 0.78 µg/ml were shortlisted for secondary and tertiary screening.Further shortlisting was done from these rounds, to select the top 3 compounds with MIC ranging between 1.56 and 0.78 µg/ml.Since only three shortlisted hits consistently showed potent antimycobacterial activity, they were then tested more rigorously using a variety of methods discussed in the main body of the manuscript.
The three most potent compounds identified from the above MABA screen were then tested in combination with the first-line anti-TB drugs.For this experiment, MABA was performed with minor modifications.Briefly, for each of the three potent compounds, 3-4 concentrations were typically tested, (a) at MIC (b) at two concentrations below, and (c) at one concentration above MIC in the range of 3.125 µg/ml to 0.19 µg/ml (two-fold serial dilution).The standard anti-TB drugs were tested in the range of 0.19 µg/ml to 0.006 µg/ml (two-fold serial dilution) for rifampicin and 0.39 µg/ml to 0.012 µg/ml (two-fold serial dilution) for isoniazid.To achieve this, 100 µl of first-line anti-TB drug at 2× of desired concentrations were added to the wells, followed by the addition of 50 µl of 4× concentration of the short-listed compounds.Following the addition of compounds, bacterial culture was diluted to obtain an OD 600nm of 0.02, and 50 µl was added to each well to obtain a final volume of 200 µl/well containing ~ 10 5 CFU/well.The plate was then sealed and incubated at 37 °C for 6 days, followed by Alamar Blue addition and analysis of results as described above.The fractional inhibitory concentration index (FICI) was calculated to determine the synergistic effect of these compounds with rifampicin and isoniazid as described 45 .The fractional inhibitory concentration for each compound was calculated as: FICI was calculated as FIC A + FIC B. Synergism is defined as FICI ≤ 0.5, antagonism as FICI ≥ 4 and no interaction if the FICI values range from 0.5-4.

Determination of cytotoxicity of shortlisted compounds by MTT assay
To determine the cytotoxicity of the shortlisted compounds, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed as per manufacturer's instructions.This assay allows the measurement of cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity.Briefly, HEK293t cell line (human embryonic kidney cell line) was maintained in high glucose Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/l glucose, 2 mM l-glutamine, 25 mM HEPES buffer, and 3.7 g/l sodium bicarbonate (Himedia) supplemented with 10% fetal bovine serum and 1× Pen Strep (penicillin and streptomycin solution) at 37 °C and 5% CO 2. In a 96-well plate, 2 × 10 4 cells were seeded and allowed to get adhered to the dish for 12 h.Following, adherence, all the media was replaced with 100 µl of fresh media (without antibiotic).In a separate 96-well plate, the three shortlisted compounds were twofold serially diluted in complete DMEM (without antibiotics).Diluted compounds were then added to the cells at a final concentration of 50 µg/ml to 0.78 µg/ ml.The plate was then incubated for 48 h at 37 °C and 5% CO 2 .On Day 4, a stock solution of MTT (Sigma) was freshly prepared in sterile 1× PBS (Himedia) at a concentration of 20 mg/ml.MTT was further diluted to 5 mg/ ml in complete DMEM (without antibiotics), followed by the addition of 20 µl of this diluted MTT into each well and incubated for 4 h.After 4 h, the media was completely removed and 100 µl of absolute DMSO was added to dissolve the insoluble formazan crystals formed by the action of mitochondrial dehydrogenase of live cells on soluble MTT (yellow).After two hours of the dissolution of formazan crystals, purple coloured solution was analysed by taking absorbance using a microplate (ELISA) reader at 570 nm and 620 nm using M4 SpectraMax ® (Molecular Devices LLC).Deeper the purple color, the greater is the cell viability.Percentage cytotoxicity was calculated as described previously, using the following formula: Shortlisted compounds were tested for their intracellular anti-mycobacterial activity by methods as described previously 46 with minor modification.Briefly, the THP-1 cell line (human monocytic leukaemia cell line) was maintained in RPMI 1640 with 2 mM l-glutamine, 25 mM HEPES buffer, and 2 g/l sodium bicarbonate (Himedia) supplemented with 10% fetal bovine serum and 1× Pen Strep (Penicillin and streptomycin solution) at 37 °C and 5% CO 2. In a 48-well plate (Thermo Scientific™ Nunc), 3 × 10 4 THP-1 cells were seeded and differentiated by the addition of 5 ng/ml PMA (Sigma) in complete RPMI media (Himedia) (with 1× Pen Strep) as described above.Differentiated macrophages were then infected with M. tb.(H37Rv) at a multiplicity of infection (MOI) of 1:5 (1 macrophage: 5 bacteria) for 4 h 47 .Extracellular non-phagocytosed bacteria were removed by washing the infected cells three times with warm DPBS.For day 0 count, we lysed the infected macrophages (n = 3) to determine the CFU by plating various dilutions of the lysates on 7H11 agar and incubated at 37 °C for 3-4 weeks followed by bacterial enumeration and calculations.At this point we observed ~ 10,000 CFU/ml (per well).This reveals an actual MOI of 1: 0.33 equating to ~ 3 M. tb per 10 macrophage cells.For calculating the impact of drugs on growth inhibition of M. tb in macrophages, we first infected the macrophages in similar fashion as described above.Following removal of extracellular bacteria post 4 h infection, the shortlisted compounds or first-line anti-TB drugs were prepared as stock containing 5× of the desired concentration in incomplete RPMI media (without FBS and without Pen Strep).For treatment, DPBS was removed and 400 µl of incomplete media (without FBS and without Pen strep) was added to each well, followed by the addition of 100 µl of first-line anti-TB drug or short-listed compounds at 5× of the desired concentrations, to obtain a final volume of 500 µl/well.Plates were then incubated at 37 °C and 5% CO 2 for 5 days.On day 6, we collected supernatant and mixed with the macrophages lysed with lysis buffer (sterile 0.025% SDS), followed by centrifugation to pellet down all the bacilli.Following this step, bacteria is then plated on to 7H11 agar plates.The plates were incubated at 37 °C for 3-4 weeks and colonies were counted for CFU determination.

Determination of anti-mycobacterial activity of drugs using nutrient starvation model of dormancy by most probable number (MPN) method
Shortlisted compounds were tested for their anti-mycobacterial activity employing nutrient starvation dormancy model using MPN assay as described previously 48 and in Supplementary Fig. 5.To mimic the dormant conditions, M. tb H37Rv was first grown in nutrient rich (7H9 supplemented with OADC and 0.05% tween 80) media for 7 days until an OD 600nm of 0.4-0.8.On day 8, the bacterial culture was centrifuged, washed twice with sterile PBS (Himedia) and then re-suspended in 10 mL PBS in sealed tubes.The sealed tubes were incubated at 37 °C with 5% CO 2 in humid and stationary condition (without shaking) for 6 weeks.Following 6 weeks of starvation, 200 µl of nutrient-starved cultures were taken in a microfuge-tubes and treated with 10 µM of the first-line TB drugs, rifampicin and isoniazid and three of our shortlisted compounds for 7 days at 37 °C with 5% CO 2 in humid and stationary condition (without shaking).Untreated starved cells were similarly incubated as a control group.Post 7 weeks of starvation (including 1 week of drug treatment) cells were ten-fold serially diluted (10 to10 6 ) in complete 7H9 media in microfuge tubes.From each serial dilution, 50 µl of diluted cultures were plated into a 48-well plate in triplicates in a 450 µl of 7H9 (complete) making a total volume of 500 µl/well.The plates were then incubated at 37 °C with 5% CO 2 in humid and stationary condition for 2-3 weeks (until bacterial growth is observed in most diluted untreated culture).Following this, MPN of bacterial cells was calculated.Briefly, as described in Supplementary Fig. 5, bacterial growth in three consecutive dilutions (10 -5 , 10 -6 and 10 -7 ) was noted.M. tb viability was calculated as mean MPN/ml.The data is represented as the mean of n = 3 biological replicates with 95% confidence interval as per the three-tube MPN table 48 .The MPN methods have recently been applied for preclinical evaluation of anti-TB drugs using mouse tissues as well 49 .

Statistical analysis
Data was analysed using Microsoft excel, GraphPad Prism Software Version 5.0 using Student's t-test and oneway ANOVA, wherever applicable.

Figure 1 .
Figure 1.Validation of RibH as a potential drug target using CRISPRi.(A) CRISPRi design strategy.ribH gene (Rv1416) specific complementary oligonucleotides were designed and cloned as described in methods.The resulting strain was treated with anhydrotetracycline (ATc).(B) Quantitative RT-PCR to validate CRISPRimediated silencing of ribH.RT-PCR analysis was carried out showing reduced levels of ribH transcripts in ribH-KD in comparison to the parent M. tb mc 2 7902 at 50 and 100 ng/ml ATc.Fold change data (2 −ddCT ; n = 4) is plotted with respect to the parent strain.Fold change value is represented as mean ± s.d and statistical significance was measured using Student's unpaired t-test, ns non-significant, ****p < 0.0001.(C) Time-stamped growth curves of parent mc 2 7902 and ribH-KD at different concentrations of ATc (0, 50 and 100 ng/ml), ribH-KD shows delayed and reduced growth in experiments 1 and 2.

Figure 2 .
Figure 2. Structural characterization of M. tb RibH.(A) Surface and cartoon representation of one subunit of M. tb RibH depicting the secondary structure elements from the N-terminus to the C-terminus, (B) schematic representation of the DMRL (6,7-dimethyl-8-ribityllumazine synthase) domain (upper panel) and secondary structure organization of M. tb RibH that belongs to α/β/α sandwich topology, where alpha helices are represented as pipes, beta strands are represented as arrows (bottom panel) and (C) the homopentameric M. tb RibH (PDB ID 2C92) is shown as a cartoon with its active site present at the interface of two chains.Overall view of the M. tb RibH-riboflavin complex where the active site is occupied by riboflavin is shown, (D) surface representation of the M. tb RibH bound to riboflavin. Figure illustrates the substrate binding groove (cavity volume 889 Å 3 ).The zoomed view shows the surface of the binding pocket with riboflavin buried inside.

Figure 3 .
Figure 3. Anti-mycobacterial activity of shortlisted RibH inhibitors.The figure depicts the representative pictograms obtained on screening of anti-mycobacterial activity of three of the shortlisted compounds namely, A10, A39 and A52 against M. tb H37Rv by microplate Alamar blue assay (MABA).The assay was performed at least three times with three technical replicates in each case.Rifampicin and shortlisted compounds were tested at various concentrations between 50 and 0.09 µg/ml (two-fold serial dilution).In our assay rifampicin exhibited an MIC of 0.09-0.048µg/ml.(A) Compound A10 exhibited an MIC of 1.56 µg/ml, (B) compound A39 exhibited an MIC of 3.125-1.56µg/ml, (C) compound A52 exhibited an MIC of 0.78 µg/ml.(D) Tabulation of MIC values obtained for various compounds with respect to rifampicin.The experiments were performed at three times with three technical replicates.

Figure 5 .
Figure 5. Microscale thermophoresis (MST) analysis of RibH protein with riboflavin and shortlisted drug molecules.Binding affinity of riboflavin and shortlisted drug molecules to RibH purified protein was determined by studying protein-ligand interaction using 16 concentrations (500-0.015nM) of riboflavin or shortlisted drug molecules namely, A10, A39 and A52 with a fixed concentration of fluorescently labelled M. tb RibH protein (50 nM).MST traces corresponding to various concentrations are illustrated for, (A) Riboflavin, (B) A10, (C) A39 and (D) A52.Regression curves were generated to determine the bound fraction of the ligands, thereby calculating the binding affinity as K d values.Binding affinity were determined to be (E) 86 nM for riboflavin, (F) 69 nM for A10, (G) 2.3 nM for A39 and (H) 48 nM for A52.The data for three independent experiments (n = 3) is presented as mean ± s.d.

Figure 8 .
Figure 8. RibH targeting drugs interfere with growth (resuscitation) of nutritionally starved M. tb in an in vitro dormancy model.M. tb.H37Rv cultures were starved for 6 weeks in phosphate buffered saline, followed by treatment with the compounds namely, A10, A39 and A52 or first-line anti-TB drugs isoniazid or rifampicin at an equimolar concentration of 10 µM.Bacterial count estimation was carried out using the MPN (most probable number) assay.CFU data (n = 3) is represented as geometric mean ± SEM log 10 CFU/ml.Data represents geometric mean with 95% CI for (n = 3).All the three compounds gave significant growth inhibition as compared to untreated control (p < 0.0001, two-tailed t-test using GraphPad Prism software).Compound A10 shows significant reduction in M. tb growth as compared to the first line anti-TB drug Isoniazid (p < 0.05, oneway ANOVA using GraphPad Prism software). https://doi.org/10.1038/s41598-024-63051-6 https://doi.org/10.1038/s41598-024-63051-6

Fractional
inhibitory concentration of compound A : FIC A = (MIC of compound A in the presence of compound B)/(MIC of compound A alone); Fractional inhibitory concentration of compound B : FIC B = (MIC of compound B in the presence of compound A/(MIC of compound B alone); Vol.:(0123456789) Scientific Reports | (2024) 14:12170 | https://doi.org/10.1038/s41598-024-63051-6www.nature.com/scientificreports/Determination of intracellular anti-mycobacterial activity of the shortlisted compounds in the THP-1-derived macrophages

Determination of minimal inhibitory concentration (MIC) of shortlisted compounds against M. tb by microplate Alamar Blue assay (MABA)
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