Repurposing of conformationally-restricted cyclopentane-based AKT-inhibitors leads to discovery of potential and more selective antileishmanial agents than miltefosine.

Conformational restriction was addressed towards the development of more selective and effective antileishmanial agents than currently used drugs for treatment of Leishmania donovani; the causative parasite of the fatal visceral leishmaniasis. Five types of cyclopentane-based conformationally restricted miltefosine analogs that were previously explored in literature as anticancer AKT-inhibitors were reprepared and repurposed as antileishmanial agents. Amongst, positions-1 and 2 cis-conformationally-restricted compound 1a and positions-2 and 3 trans-conformationally-restricted compound 3b were highly potent eliciting sub-micromolar IC50 values for inhibition of infection and inhibition of parasite number compared with the currently used miltefosine drug that showed low micromolar IC50 values for inhibition of infection and inhibition of parasite number. Compounds 1a and 3b eradicated the parasite without triggering host cells cytotoxicity over more than one log concentration interval which is a superior performance compared to miltefosine. In silico studies suggested that conformational restriction conserved the conformer capable of binding LdAKT-like kinase while it might be possible that it excludes other conformers mediating undesirable effects and/or toxicity of miltefosine. Together, this study presents compounds 1a and 3b as antileishmanial agents with superior performance over the currently used miltefosine drug.

Synthesis, In Silico and In Vitro Characterization of Novel N,N-Substituted Pyrazolopyrimidine Acetamide Derivatives for the 18KDa Translocator Protein (TSPO).

The translocator protein (TSPO) is an interesting biological target for molecular imaging and therapy because the overexpression of TSPO is associated with microglial activation caused by neuronal damage or neuroinflammation, and these activated microglia are involved in various central nervous system (CNS) diseases. The TSPO is a target for neuroprotective treatment, which is used with the aim of reducing microglial cell activation. The novel N,N-disubstituted pyrazolopyrimidine acetamides scaffold (GMA 7-17), which bears a fluorine atom and is directly linked to the phenyl moiety, was synthesized, and each of the novel ligands was characterized in vitro. All of the newly synthesized ligands displayed picomolar to nanomolar affinity for the TSPO. Particularly, an in vitro affinity study led to the discovery of 2-(5,7-diethyl-2-(4-fluorophenyl)pyrazolo [1,5-a]pyrimidin-3-yl)-N-ethyl-N-phenylacetamide GMA 15 (Ki = 60 pM), a novel TSPO ligand that exhibits a 61-fold enhancement in affinity compared to the reference standard DPA-714 (Ki = 3.66 nM). Molecular dynamic (MD) studies of the highest affinity binder, GMA 15, were carried out to check its time-dependent stability with the receptor compared to DPA-714 and PK11195. The hydrogen bond plot also indicated that GMA 15 formed higher hydrogen bonds compared to DPA-714 and PK11195. We anticipate that further optimization to enhance the potency in a cellular assay needs to be followed, but our strategy of identifying potential TSPO binding novel scaffolds may open up a new avenue to develop novel TSPO ligands suited for potential molecular imaging and a wide range of therapeutic applications.

Bioinformatic Analyses of Canonical Pathways of TSPOAP1 and its Roles in Human Diseases.

TSPO-associated protein 1 (TSPOAP1) is a cytoplasmic protein and is closely associated with its mitochondrial transmembrane protein partner translocator protein (TSPO). To decipher the canonical signalling pathways of TSPOAP1, its role in human diseases and disorders, and relationship with TSPO; expression analyses of TSPOAP1- and TSPO-associated human genes were performed by Qiagen Ingenuity Pathway Analysis (IPA). In the expression analysis, necroptosis and sirtuin signalling pathways, mitochondrial dysfunction, and inflammasome were the top canonical pathways for both TSPOAP1 and TSPO, confirming the close relationship between these two proteins. A distribution analysis of common proteins in all the canonical pathways predicted for TSPOAP1 revealed that tumor necrosis factor receptor 1 (TNFR1), vascular cell adhesion molecule 1 (VCAM1), cyclic AMP response element-binding protein 1 (CREB1), T-cell receptor (TCR), nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3), DNA-dependent protein kinase (DNA-PK or PRKDC), and mitochondrial permeability transition pore (mPTP) were the major interaction partners of TSPOAP1, highlighting the role of TSPOAP1 in inflammation, particularly neuroinflammation. An analysis of the overlap between TSPO and TSPOAP1 Homo sapiens genes and top-ranked canonical pathways indicated that TSPO and TSPOAP1 interact via voltage-dependent anion-selective channels (VDAC1/2/3). A heat map analysis indicated that TSPOAP1 has critical roles in inflammatory, neuroinflammatory, psychiatric, and metabolic diseases and disorders, and cancer. Taken together, this information improves our understanding of the mechanism of action and biological functions of TSPOAP1 as well as its relationship with TSPO; furthermore, these results could provide new directions for in-depth functional studies of TSPOAP1 aimed at unmasking its detailed functions.