Diógenes Santiago Santos

The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K _m , k _cat , and k _cat /K _m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k _cat /K _m for NADH and k _cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E _a and E _η ), enthalpy (∆H ^# ), entropy (∆S ^# ), and Gibbs free energy (∆G ^# ) of activation were determined. The ∆G ^# values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES ^# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that “soft” interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.

A ultrasound-assisted oxidative cyclization of semicarbazones using N-bromosuccinimide in the presence of sodium acetate was established providing efficient and rapid access to a variety of 2-amino-1,3,4-oxadiazoles. Moreover, the new synthetic protocol provides a simple procedure utilizing a safer oxidizing system that affords the target products in high regioselectivity, satisfactory yields, and elevated purities. GRAPHICAL ABSTRACT

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Key words: iunH gene; nucleoside hydrolase; gene knockout; Mycobacterium tuberculosis

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis.

In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.

HisD-Encoded histidinol dehydrogenase (HisD) catalyzes the two last chemical reactions of the L-histidine biosynthetic pathway, namely the conversion of L-histidinol (L-Hol) to L-histidinaldehyde (L-Hal) and to L-histidine (L-His). The hisD gene product has been shown to be essential for Mycobacterium tuberculosis survival in vitro. Herein, we describe a series of biochemical studies on recombinant Mycobacterium tuberculosis HisD (MtHisD). The synthesis of hydrazones derived from L-histidine yielded inhibitors in the low micromolar range, one of which showed moderate anti-Mtb activity. The compounds described here are, to the best of our knowledge, the first inhibitors of MtHisD activity reported in the literature, and they could become promising candidates for future development.