Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence.Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy –performed in a large and diverse chemolibrary– complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening.Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12–20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7–45 μM).Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known “garbage in, garbage out” machine learning principle

Neuroprotective effects of steroid analogues on P19-N neurons

Naturally occurring neurosteroids are potent allosteric modulators of gamma-aminobutyric acid(A) receptor and through augmentation of gamma-aminobutyric acid(A) receptor function, can protect neuronal cells against N-methyl-D-aspartate receptor over-activation, ischemia and traumatic brain injury. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons and were subjected to excitotoxicity in the presence of N-methyl-D-aspartate. Novel synthetic analogues of the endogenous neurosteroids allopregnanolone and dehydroepiandrostrone, inhibited excitotoxic cell death of P19-N neurons, by directly maintaining the activation of PKB/Akt kinase, and interfering with the intrinsic mitochondrial apoptotic pathway, preserving cytochrome c in the mitochondria and Bax in the cytoplasm. The efficiency and the potency of these neurosteroids were similar to those of allopregnanolone and dehydroepiandrostrone. Their effects were gamma-aminobutyric acid(A) receptor mediated, since they were abolished in the presence of bicuculline, an antagonist of receptor’s function. In addition, the synthetic compounds retained the ability to alter gamma-aminobutyric acid(A) receptor subunit gene expression, but their effects on transcriptional activity were less pronounced than those of allopregnanolone and dehydroepiandrostrone. These results suggest that synthetic neurosteroids may serve as potent, membrane acting, neuroprotectants against N-methyl-D-aspartate receptor neurotoxicity on neuronal cells. (c) 2007 Elsevier Ltd. All rights reserved

A 17 beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA(A) receptors with nanomolar affinity

Journal URL: http://www.nature.com/bjp/index.htmlThis is also freely available in PubMed Central at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2042931

Synergistic combination of alkylphosphocholines with peptaibols in targeting Leishmania infantum in vitro

Anti-leishmanial treatment increasingly encounters therapeutic limitations due to drug toxicity and development of resistance. The effort for new therapeutic strategies led us to work on combinations of chemically different compounds that could yield enhanced leishmanicidal effect. Peptaibols are a special type of antimicrobial peptides that are able to form ion channels in cell membranes and potentially affect cell viability. We assayed the antileishmanial activity of two well studied helical peptaibols, the 16-residue antiamoebin and the 20-residue alamethicin-analogue suzukacillin, and we evaluated the biological effect of their combination with the alkylphosphocholine miltefosine and its synthetic analogue TC52. The peptaibols tested exhibited only moderate antileishmanial activity, however their combination with miltefosine had a super-additive effect against the intracellular parasite (combination index 0.83 and 0.43 for antiamoebin and suzukacillin respectively). Drug combinations altered the redox stage of promastigotes, rapidly dissipated mitochondrial membrane potential and induced concatenation of mitochondrial network promoting spheroidal morphology. These results evidenced a potent and specific antileishmanial effect of the peptaibols/miltefosine combinations, achieved with significantly lower concentrations of the compounds compared to monotherapy. Furthermore, they revealed the importance of exploring novel classes of bioactive compounds such as peptaibols and demonstrated for the first time that they can act in synergy with currently used antileishmanial drugs to improve the therapeutic outcome. Keywords: Leishmaniasis therapy, Miltefosine, Peptaibol antibiotics, Drug synergy, Mitochondrial membrane potential, Reactive oxygen specie