N-Acyl phenothiazines as mycobacterial ATP synthase inhibitors: Rational design, synthesis and in vitro evaluation against drug sensitive, RR and MDR-TB.

The mycobacterial F-ATP synthase is responsible for the optimal growth, metabolism and viability of Mycobacteria, establishing it as a validated target for the development of anti-TB therapeutics. Herein, we report the discovery of an N-acyl phenothiazine derivative, termed PT6, targeting the mycobacterial F-ATP synthase. PT6 is bactericidal and active against the drug sensitive, Rifampicin-resistant as well as Multidrug-resistant tuberculosis strains. Compound PT6 showed noteworthy inhibition of F-ATP synthesis, exhibiting an IC50 of 0.788 µM in M. smegmatis IMVs and was observed that it could deplete intracellular ATP levels, exhibiting an IC50 of 30 µM. PT6 displayed a high selectivity towards mycobacterial ATP synthase compared to mitochondrial ATP synthase. Compound PT6 showed a minor synergistic effect in combination with Rifampicin and Isoniazid. PT6 demonstrated null cytotoxicity as confirmed by assessing its toxicity against VERO cell lines. Further, the binding mechanism and the activity profile of PT6 were validated by employing in silico techniques such as molecular docking, Prime MM/GBSA, DFT and ADMET analysis. These results suggest that PT6 presents an attractive lead for the discovery of a novel class of mycobacterial F-ATP synthase inhibitors.

Dual transcriptional inhibition of glutamate and alanine racemase is synergistic in Mycobacterium tuberculosis.

Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis. This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.

Identification of antimycobacterial 8-hydroxyquinoline derivatives as in vitro enzymatic inhibitors of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase.

The increasing prevalence of drug-resistant Mycobacterium tuberculosis strains stimulates the discovery of new drug candidates. Among them are 8-hydroxyquinoline (8HQ) derivatives that exhibited antimicrobial properties. Unfortunately, there is a lack of data assessing possible targets for this class mainly against Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (MtInhA), a validated target in this field. Thus, the main purpose of this study was to identify 8HQ derivatives that are active against M. tuberculosis and MtInhA. Initially, the screening against the microorganism of a small antimicrobial library and its new derivatives that possess some structural similarity with MtInhA inhibitors identified four 7-substituted-8HQ (series 5 - 5a, 5c, 5d and 5i) and four 5-substituted-8HQ active derivatives (series 7 - 7a, 7c, 7d and 7j). In general, the 7-substituted 8-HQs were more potent and, in the enzymatic assay, were able to inhibit MtInhA at low micromolar range. However, the 5-substituted-8-HQs that presented antimycobacterial activity were not able to inhibit MtInhA. These findings indicate the non-promiscuous nature of 8-HQ derivatives and emphasize the significance of selecting appropriate substituents to achieve in vitro enzyme inhibition. Finally, 7-substituted-8HQ series are promising new derivatives for structure-based drug design and further development.

Crystal structure of dihydroneopterin aldolase from Mycobacterium tuberculosis associated with 8-mercaptoguanine, and development of novel S8-functionalized analogues as inhibitors: Synthesis, enzyme inhibition, in vitro toxicity and antitubercular activity.

The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis (MtFolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of MtFolB. These compounds exhibited IC50 values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting MtFolB, an attractive molecular target for TB drug development.

Identification of kinase inhibitors as potential host-directed therapies for intracellular bacteria.

The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competitive kinase inhibitors with known target profiles from two Published Kinase Inhibitor Sets (PKIS1 and PKIS2) using intracellular infection models for Stm and Mtb, based on human cell lines and primary macrophages. Additionally, the in vivo safety and efficacy of the compounds were assessed using zebrafish embryo infection models. Our screen identified 11 hit compounds for Stm and 17 hit compounds for Mtb that were effective against intracellular bacteria and non-toxic for host cells. Further experiments were conducted to prioritize Stm hit compounds that were able to clear the intracellular infection in primary human macrophages. From these, two structurally related Stm hit compounds, GSK1379738A and GSK1379760A, exhibited significant activity against Stm in infected zebrafish embryos. In addition, we identified compounds that were active against intracellular Mtb, including morpholino-imidazo/triazolo-pyrimidinones that target PIK3CB, as well as 2-aminobenzimidazoles targeting ABL1. Overall, this study provided insights into kinase targets acting at the host-pathogen interface and identified several kinase inhibitors as potential HDTs.

Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 µM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 µg/mL, respectively, superior to novobiocin (MIC > 100.0 µg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

Crystal structure and function analysis of 6-phosphogluconate dehydrogenase in Mycobacterium tuberculosis.

6-phosphogluconate dehydrogenase (6PGDH) is an essential enzyme in energy metabolism and redox reactions, and represents a potential drug target for the development of therapies targeting trypanosomes, plasmodium, or other pathogens. Tuberculosis, caused by Mycobacterium tuberculosis, is a contagious disease that severely affects human health, with approximately one-third of the world's population infected. However, the protein structure, exact oligomeric state, and catalytic mechanism of 6PGDH in Mycobacterium tuberculosis (Mt6PGDH) have remained largely unknown. In this study, we successfully purified and determined the structure of Mt6PGDH, revealing its function as a tetramer in both solution and crystal states. Through structural comparisons, we clarified the tetramer formation mechanism and the oligomeric organization of short-chain 6PGDHs. Additionally, we identified key residues for coenzyme recognition and catalytic activity. This work not only deepens our understanding of the enzymatic function of Mt6PGDH but also lays a foundation for the development of drugs targeting this enzyme.

Identification and Design of Novel Potential Antimicrobial Peptides Targeting Mycobacterial Protein Kinase PknB.

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site's intrinsic dynamic movement to estimate the new peptide's role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.

Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587.

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.

Chemoenzymatic Synthesis of Fluorinated Mycocyclosin Enabled by the Engineered Cytochrome P450-Catalyzed Biaryl Coupling Reaction.

Installing fluorine atoms onto natural products holds great promise for the generation of fluorinated molecules with improved or novel pharmacological properties. The enzymatic oxidative carbon-carbon coupling reaction represents a straightforward strategy for synthesizing biaryl architectures, but the exploration of this method for producing fluorine-substituted derivatives of natural products remains elusive. Here, in this study, we report the protein engineering of cytochrome P450 from Mycobacterium tuberculosis (MtCYP121) for the construction of a series of new-to-nature fluorine-substituted Mycocyclosin derivatives. This protocol takes advantage of a "hybrid" chemoenzymatic procedure consisting of tyrosine phenol lyase-catalyzed fluorotyrosine preparation from commercially available fluorophenols, intermolecular chemical condensation to give cyclodityrosines, and an engineered MtCYP121-catalyzed intramolecular biphenol coupling reaction to complete the strained macrocyclic structure. Computational mechanistic studies reveal that MtCYP121 employs Cpd I to abstract a hydrogen atom from the proximal phenolic hydroxyl group of the substrate to trigger the reaction. Then, conformational change makes the two phenolic hydroxyl groups close enough to undergo intramolecular hydrogen atom transfer with the assistance of a pocket water molecule. The final diradical coupling process completes the intramolecular C-C bond formation. The efficiency of the biaryl coupling reaction was found to be influenced by various fluorine substitutions, primarily due to the presence of distinct binding conformations.

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

A Noble Metal Substitution Leads to B12 Cofactor Mimicry by a Rhodibalamin.

In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.

New coumarin linked thiazole derivatives as antimycobacterial agents: Design, synthesis, enoyl acyl carrier protein reductase (InhA) inhibition and molecular modeling.

Tuberculosis is a global serious problem that imposes major health, economic and social challenges worldwide. The search for new antitubercular drugs is extremely important which could be achieved via inhibition of different druggable targets. Mycobacterium tuberculosis enoyl acyl carrier protein reductase (InhA) enzyme is essential for the survival of M. tuberculosis. In this investigation, a series of coumarin based thiazole derivatives was synthesized relying on a molecular hybridization approach and was assessed against thewild typeMtb H37Rv and its mutant strain (ΔkatG) via inhibiting InhA enzyme. Among the synthesized derivatives, compounds 2b, 3i and 3j were the most potent against wild type M. tuberculosis with MIC values ranging from 6 to 8 µg/ mL and displayed low cytotoxicity towards mouse fibroblasts at concentrations 8-13 times higher than the MIC values. The three hybrids could also inhibit the growth of ΔkatGmutant strain which is resistant to isoniazid (INH). Compounds 2b and 3j were able to inhibit the growth of mycobacteria inside human macrophages, indicating their ability to penetrate human professional phagocytes. The two derivatives significantly suppress mycobacterial biofilm formation by 10-15 %. The promising target compounds were also assessed for their inhibitory effect against InhA and showed potent effectiveness with IC50 values of 0.737 and 1.494 µM, respectively. Molecular docking studies revealed that the tested compounds occupied the active site of InhA in contact with the NAD+ molecule. The 4-phenylcoumarin aromatic system showed binding interactions within the hydrophobic pocket of the active site. Furthermore, H-bond formation and π -π stacking interactions were also recorded for the promising derivatives.

Identification of potent indolizine derivatives against Mycobacterial tuberculosis: In vitro anti-TB properties, in silico target validation, molecular docking and dynamics studies.

In the current study, two sets of compounds: (E)-1-(2-(4-substitutedphenyl)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium derivatives (3a-3e); and (E)-3-(substitutedbenzoyl)-7-((hydroxyimino)methyl)-2-substitutedindolizine-1-carboxylate derivatives (5a-5j), were synthesized and biologically evaluated against two strains of Mycobacterial tuberculosis (ATCC 25177) and multi-drug resistant (MDR) strains. Further, they were also tested in vitro against the mycobacterial InhA enzyme. The in vitro results showed excellent inhibitory activities against both MTB strains and compounds 5a-5j were found to be more potent, and their MIC values ranged from 5 to 16 µg/mL and 16-64 µg/mL against the M. tuberculosis (ATCC 25177) and MDR-TB strains, respectively. Compound 5h with phenyl and 4-fluorobenzoyl groups attached to the 2- and 3-position of the indolizine core was found to be the most active against both strains with MIC values of 5 µg/mL and 16 µg/mL, respectively. On the other hand, the two sets of compounds showed weak to moderate inhibition of InhA enzyme activity that ranged from 5 to 17 % and 10-52 %, respectively, with compound 5f containing 4-fluoro benzoyl group attached to the 3-position of the indolizine core being the most active (52 % inhibition of InhA). Unfortunately, there was no clear correlation between the InhA inhibitory activity and MIC values of the tested compounds, indicating the probability that they might have different modes of action other than InhA inhibition. Therefore, a computational investigation was conducted by employing molecular docking to identify their putative drug target(s) and, consequently, understand their mechanism of action. A panel of 20 essential mycobacterial enzymes was investigated, of which ß-ketoacyl acyl carrier protein synthase I (KasA) and pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (BioA) enzymes were revealed as putative targets for compounds 3a-3e and 5a-5j, respectively. Moreover, in silico ADMET predictions showed adequate properties for these compounds, making them promising leads worthy of further optimization.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Screening Potential Citrate Lyase Inhibitors from a Library of Marine Compounds.

Tuberculosis, a persistent illness caused by Mycobacterium tuberculosis, remains a significant global public health challenge. The widespread use of anti-tuberculosis drugs has resulted in the emergence of drug-resistant strains, which complicates treatment efforts. Addressing this issue is crucial and hinges on the development of new drugs that can effectively target the disease. This involves identifying novel therapeutic targets that can disrupt the bacterium's survival mechanisms in various environments such as granulomas and lesions. Citrate lyase, essential for the survival of Mycobacterium species at lesion sites and in granulomatous conditions, is a potential target for the treatment of tuberculosis. This manuscript aimed to construct an efficient enzyme inhibitor screening platform using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). This system can accurately identify compounds with enzyme inhibitory activity from a library of marine terpenoids and phenolic compounds. Utilizing the screened herbal enzyme inhibitors as a starting point, we analyzed their chemical structures and skillfully built a library of marine compounds based on these structures. The results showed that all of the tested compounds from the phenolics library inhibited citrate lyase by more than 50%, and a significant portion of terpenoids also demonstrated inhibition, with these active terpenoids comprising over half of the terpenoids tested. The study underscores the potential of marine-derived phenolic and terpenoid compounds as potent inhibitors of citrate lyase, indicating a promising direction for future investigations in treating tuberculosis and associated disorders.

A consensus reverse docking approach for identification of a competitive inhibitor of acetyltransferase enhanced intracellular survival protein from Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.

Computational Insights into the Intramolecular Aromatic C-C Coupling Catalyzed by the Cytochrome P450 Enzyme CYP121 from Mycobacterium tuberculosis.

CYP121 is a P450 enzyme that catalyzes the intramolecular C-C coupling of its native substrate, dicyclotyrosine (cYY). According to previous suggestions, when the cosubstrate peracetic acid was used to generate Cpd I, the substrate cYY was suggested to participate in the cleavage of the O-O bond; however, whether cYY is involved in the formation of Cpd I and how two distant aromatic carbon atoms are activated are still unclear. Here, we constructed computational models and performed QM/MM calculations to clarify the reaction mechanism. On the basis of our calculation results, cYY is not involved in the formation of Cpd I, and the C-C coupling reaction starts from hydrogen abstraction. In the second stage, the substrate should first undergo a complex conformational change, leading to two phenolic hydroxyls of cYY close to each other. In the subsequent reaction, the resultant Cpd II again abstracts a hydrogen atom from the proximal tyrosine to generate the diradical intermediate. In addition, the C-C coupling occurs in the active site, but the final aromatization may be a nonenzymatic reaction. In general, the intramolecular C-C coupling requires two basic conditions, including the active site having good flexibility and the substrate itself having a suitable and rotatable skeleton.

Evolution of fungal tuberculosis necrotizing toxin (TNT) domain-containing enzymes reveals divergent adaptations to enhance NAD cleavage.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.