Identification of potential inhibitors of Mycobacterium tuberculosis shikimate kinase: molecular docking, in silico toxicity and in vitro experiments.

Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb.

Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity, SAR, and preclinical evaluation.

Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals, satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-aminoquinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-resistant tuberculosis treatments.

Antitubercular Activity of Novel 2-(Quinoline-4-yloxy)acetamides with Improved Drug-Like Properties.

Using cycloalkyl and electron-donating groups to decrease the carbonyl electrophilicity, a novel series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Structure-activity relationship studies led to selective and potent antitubercular agents with minimum inhibitory concentrations in the submicromolar range against drug-sensitive and drug-resistant Mtb strains. An evaluation of the activity of the lead compounds against a spontaneous qcrB mutant strain indicated that the structures targeted the cytochrome bc 1 complex. In addition, selected molecules inhibited Mtb growth in a macrophage model of tuberculosis infection. Furthermore, the leading compound was chemically stable depending on the context and showed good kinetic solubility, high permeability, and a low rate of in vitro metabolism. Finally, the pharmacokinetic profile of the compound was assessed after oral administration to mice. To the best of our knowledge, for the first time, a 2-(quinoline-4-yloxy)acetamide was obtained with a sufficient exposure, which may enable in vivo effectiveness and its further development as an antituberculosis drug candidate.

Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate.