Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species.

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases' participation in protein-protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment.

BACKGROUND: Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported. CASE PRESENTATION: In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy. CONCLUSIONS: This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.