Kenia Pissinate

2-(Quinolin-4-yloxy)acetamides Are Active against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains.

2-(Quinolin-4-yloxy)acetamides have been described as potent in vitro inhibitors of Mycobacterium tuberculosis growth. Herein, additional chemical modifications of lead compounds were carried out, yielding highly potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.05 μM. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to Vero and HaCat cells (IC50s ≥ 20 μM). In addition, the 2-(quinolin-4-yloxy)acetamides showed intracellular activity against the bacilli in infected macrophages with action similar to rifampin, low risk of drug-drug interactions, and no sign of cardiac toxicity in zebrafish (Danio rerio) at 1 and 5 μM. Therefore, these data indicate that this class of compounds may furnish candidates for future development to, hopefully, provide drug alternatives for tuberculosis treatment.

Piperazine derivatives: synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies.

The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates. MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis system, and is the bona fide target for isoniazid, the most prescribed drug for tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and screened as MtInhA inhibitors, which resulted in the identification of compounds with IC50 values in the submicromolar range. A structure-activity relationship (SAR) evaluation indicated the importance of the chemical environment surrounding the carbonyl group for inhibition. In addition, the structure of one selected compound was supported by crystallographic studies, and experimental geometrical values were compared with semi-empirical quantum chemical calculations. Furthermore, the mode of inhibition and inhibitory dissociation constants were determined for the nine most active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-CoA) substrate binding site.

Activity of 2-(quinolin-4-yloxy)acetamides in Mycobacterium tuberculosis clinical isolates and identification of their molecular target by whole-genome sequencing

The 2-(quinolin-4-yloxy)acetamides (QOAs) have been reported to be promising molecules for tuberculosis treatment. Recent studies demonstrated their potent antimycobacterial activity, biological stability and synergism with rifampicin. The identification of the molecular target is an essential step towards the development of a novel drug candidate. Here, we report the target identification of the QOAs. We found that these compounds are active against Mycobacterium tuberculosis clinical isolates resistant to isoniazid, rifampicin, ethambutol, streptomycin and ethionamide. The initial evidence that DNA gyrase might be the target of QOAs, based on high minimum inhibitory concentration (MIC) values against ofloxacin-resistant clinical isolates and structural similarities with fluoroquinolones, was discarded by experiments performed with M. tuberculosis GyrA point mutant, DNA gyrase supercoiling inhibition assay and overexpression of DNA gyrase. We selected spontaneous mutants for our lead compound 21 and observed that these strains were also resistant to all QOA derivatives. The genomes of the spontaneous mutants were sequenced, and the results revealed a single mutation in qcrB gene (T313A), which indicates that the QOAs target the cytochrome bc1 complex. The protein-compound interaction was further investigated by molecular docking. These findings reinforce the relevance of these compounds as promising candidates for the treatment of multidrug-resistant tuberculosis.

Ultrasound-assisted synthesis of 2-amino-1,3,4-oxadiazoles through NBS-mediated oxidative cyclization of semicarbazones

ABSTRACT 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

Inhibitory activity of pentacyano(isoniazid)ferrate(II), IQG-607, against promastigotes and amastigotes forms of Leishmania braziliensis

M. tuberculosis and parasites of the genus Leishmania present the type II fatty acid biosynthesis system (FASII). The pentacyano(isoniazid)ferrate(II) compound, named IQG-607, inhibits the enzyme 2-trans-enoyl-ACP(CoA) reductase from M. tuberculosis, a key component in the FASII system. Here, we aimed to evaluate the inhibitory activity of IQG-607 against promastigote and amastigote forms of Leishmania (Viannia) braziliensis isolated from patients with different clinical forms of L. braziliensis infection, including cutaneous, mucosal and disseminated leishmaniasis. Importantly, IQG-607 inhibited the proliferation of three different isolates of L. braziliensis promastigotes associated with cutaneous, mucosal and disseminated leishmaniasis. The IC50 values for IQG-607 ranged from 32 to 75 μM, for these forms. Additionally, IQG-607 treatment decreased the proliferation of intracellular amastigotes in infected macrophages, after an analysis of the percentage of infected cells and the number of intracellular parasites/100 cells. IQG-607 reduced from 58% to 98% the proliferation of L. braziliensis from cutaneous, mucosal and disseminated strains. Moreover, IQG-607 was also evaluated regarding its potential toxic profile, by using different cell lines. Cell viability of the lineages Vero, HaCat and HepG2 was significantly reduced after incubation with concentrations of IQG-607 higher than 2 mM. Importantly, IQG-607, in a concentration of 1 mM, did not induce DNA damage in HepG2 cells, when compared to the untreated control group. Future studies will confirm the mechanism of action of IQG-607 against L. braziliensis.

Validation of Mycobacterium tuberculosis dihydroneopterin aldolase as a molecular target for anti-tuberculosis drug development

An early step of target validation in antimicrobial drug discovery is to prove that a gene coding for a putative target is essential for pathogens viability. However, little attention has been paid to demonstrate the causal links between gene essentiality and a particular protein function that will be the focus of a drug discovery effort. This should be considered an important step in target validation since a growing number of proteins are found to exhibit multiple and unrelated tasks. Here, we show that the Mycobacterium tuberculosis (Mtb) folB gene is essential and that this essentiality depends on the dihydroneopterin aldolase/epimerase activities of its protein product, the FolB protein from the folate biosynthesis pathway. The wild-type (WT) MtFolB and point mutants K99A and Y54F were cloned, expressed, purified and monitored for the aldolase, epimerase and oxygenase activities using HPLC. In contrast to the WT MtFolB, both mutants have neither aldolase nor epimerase activities in the conditions assayed. We then performed gene knockout experiments and showed that folB gene is essential for Mtb survival under the conditions tested. Moreover, only the WT folB sequence could be used as a rescue copy in gene complementation studies. When the sequences of mutants K99A or Y54F were used for complementation, no viable colonies were obtained, indicating that aldolase and/or epimerase activities are crucial for Mtb survival. These results provide a solid basis for further work aiming to develop new anti-TB agents acting as inhibitors of the aldolase/epimerase activities of MtFolB.

Preclinical pharmacokinetic profiling of IQG-607, a potential oral metallodrug to treat tuberculosis

ABSTRACT IQG‐607 is an analog of isoniazid with anti‐tuberculosis activity. This work describes the development and validation of an HPLC method to quantify pentacyano(isoniazid)ferrate(II) compound (IQG‐607) and the pharmacokinetic studies of this compound in mice. The method showed linearity in the 0.5–50 &mgr;g/mL concentration range (r = 0.9992). Intra‐ and inter‐day precision was <5%, and the recovery ranged from 92.07 to 107.68%. IQG‐607 was stable in plasma for at least 30 days at −80 °C and, after plasma processing, for 4 h in the auto‐sampler maintained on ice (recovery >85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administration to mice. IQG‐607 levels in plasma were quantified at time points for up to 2.5 h. A short half‐life (t1/2) (1.14 h), a high clearance (CL) (3.89 L/h/kg), a moderate volume of distribution at steady state (Vdss) of 1.22 L/kg, were observed after i.v. (50 mg/kg) administration. Similar results were obtained for oral administration (250 mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87 ± 0.9%. The results described here provide novel insights into a pivotal criterion to warrant further efforts to be pursued towards attempts to translate this chemical compound into a chemotherapeutic agent to treat TB. Graphical abstract Figure. No Caption available.

Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis.

Thermodynamics, functional and structural characterization of inosine–uridine nucleoside hydrolase from Leishmania braziliensis

Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by Leishmania braziliensis. The first-line drugs used in the treatment of leishmaniasis have several limitations: parenteral administration, long duration of treatment, and serious toxicity. One key metabolic characteristic of these parasites is the lack of a de novo purine biosynthesis pathway, making them auxotrophic to purines. Accordingly, they rely solely on the purine salvage pathway for nucleotide synthesis. A better understanding of the purine salvage pathway can reveal details of the biology of L. braziliensis that could, in turn, be used to develop new strategies to combat this parasite. The inosine–uridine nucleoside hydrolase from L. braziliensis (LbIU-NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here is described cloning, heterologous protein expression, and a three-step purification protocol that yielded homogenous recombinant protein. The determination of LbIU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and p-nitrophenyl β-D-ribofuranoside is also reported. These data suggest that LbIU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests that D-ribose can bind to free enzyme with favorable enthalpic (ΔH) and entropic (ΔS) contributions. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the LbIU-NH-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, and pH-rate profiles are also presented. In addition, the crystal structure of LbIU-NH in complex with β-D-ribose and Ca2+ at 1.5 A resolution is described.

Synthesis of New trans-Dehydrocrotonin Nitrogenated Derivatives and their Cytotoxic and DNA-Topoisomerase I Inhibitory Activities

A new series of 19-nor-clerodane diterpene derivatives was synthesized from the natural trans-dehydrocrotonin obtained from stem barks of Croton cajucara (Euphorbiaceae), a native medicinal plant of the Brazilian Amazon. The new derivatives were obtained by changes in the ketone moiety of trans-dehydrocrotonin leading to nitrogenated derivatives which are: three substituted hydrazine diterpenes, oxime, and methyloxime. The cytotoxic effect of the diterpene derivatives was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma and K562 human leukemia cells. The cytotoxic activity of the hydrazine and oxime semi-synthetic derivatives was better than the one of the natural product trans-dehydrocrotonin. Moreover, all diterpenes were tested for their DNA topoisomerase I inhibitory activity, and the most effective one, in general, was observed to the phenyl-hydrazone derivative. Results indicated that the topoisomerase I inhibitory effect is correlated with the cytotoxic activity.