Identification Of Hit Compounds Research Articles

Identification of Hit Compounds Using Artificial Intelligence for the Management of Allergic Diseases.

This study aimed to identify and evaluate drug candidates targeting the kinase inhibitory region of suppressor of cytokine signaling (SOCS) 3 for the treatment of allergic rhinitis (AR). Utilizing an artificial intelligence (AI)-based new drug development platform, virtual screening was conducted to identify compounds inhibiting the SH2 domain binding of SOCS3. Luminescence assays assessed the ability of these compounds to restore JAK-2 activity diminished by SOCS3. Jurkat T and BEAS-2B cells were utilized to investigate changes in SOCS3 and STAT3 expression, along with STAT3 phosphorylation in response to the identified compounds. In an OVA-induced allergic rhinitis mouse model, we measured serum levels of total IgE and OVA-specific IgE, performed real-time PCR on nasal mucosa samples to quantify Th2 cytokines and IFN-γ expression, and conducted immunohistochemistry to analyze eosinophil levels. Screening identified 20 hit compounds with robust binding affinities. As the concentration of SOCS3 increased, a corresponding decrease in JAK2 activity was observed. Compounds 5 and 8 exhibited significant efficacy in restoring JAK2 activity without toxicity. Treatment with these compounds resulted in reduced SOCS3 expression and the reinstatement of STAT3 phosphorylation in Jurkat T and BEAS-2B cells. In the OVA-induced allergic rhinitis mouse model, compounds 5 and 8 effectively alleviated nasal symptoms and demonstrated lower levels of immune markers compared to the allergy group. This study underscores the promising nonclinical efficacy of compounds identified through the AI-based drug development platform. These findings introduce innovative strategies for the treatment of AR and highlight the potential therapeutic value of targeting SOCS3 in managing AR.

Discovery of Small-Molecule TIM-3 Inhibitors for Acute Myeloid Leukemia Using Pharmacophore-Based Virtual Screening.

T-cell immunoglobulin and mucin domain 3 (TIM-3) is a negative immune checkpoint that represents a promising target for cancer immunotherapy. Although encouraging results have been observed for TIM-3 inhibition in the context of acute myeloid leukemia (AML), targeting TIM-3 is currently restricted to monoclonal antibodies (mAbs). To fill this gap, we implemented a pharmacophore-based screening approach to identify small-molecule TIM-3 inhibitors. Our approach resulted in the identification of hit compounds with TIM-3 binding affinity. Subsequently, we used the structure-activity relationship (SAR) by a catalog approach to identify compound A-41 with submicromolar TIM-3 binding affinity. Remarkably, A-41 demonstrated the ability to block TIM-3 interactions with key ligands and inhibited the immunosuppressive function of TIM-3 using an in vitro coculture assay. This work will pave the way for future drug discovery efforts aiming at the development of small-molecule inhibitors TIM-3 for AML.

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium.

BackgroundAnthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay.Methodology/Principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3–6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug.ConclusionWith this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).

High-throughput Screening Technology for Selective Inhibitors of Transporters and Its Application in Drug Discovery

The first step in small-molecule drug discovery is the identification of hit compounds via high-throughput screening (HTS). In transporter drug discovery, most HTS assays are based on the uptake of labeled substrates, but such functional assays cannot be developed for many transporters, such as intracellular organelle transporters. These transporters remain unexplored in drug discovery despite their promise as drug targets. Affinity selection-mass spectrometry (AS-MS) is a label-free binding assay technology that has been developed as an HTS technology for analyzing interactions between targets and compounds. The use of AS-MS technology enables HTS against every type of drug target, in contrast to functional assays. AS-MS technology is usually used for soluble proteins, but we have developed this technology for application to membrane proteins as well. So far, we have used AS-MS for HTS of approximately 400000 compounds. In this review, the principles and application of AS-MS technology are introduced and an HTS campaign for solute carrier type 17A8 (SLC17A8) (vesicular glutamate transporter 3) is presented as an example.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential ‘hits’ against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.

Abstract 1738: Multiparametric approaches to evaluate mitochondrial toxicity, oxidative stress and apoptosis in drug discovery studies.

Abstract Drug discovery and toxicity studies require access to methods and technologies that can provide comprehensive evaluation of the types of cellular impacts caused by anti-cancer compounds. In particular, there is growing emphasis on the study of mitochondrial toxicity at an early phase of compound evaluation and its relations to multiple cell death processes that include apoptotic cell death, caspase-independent death and autophagic processes. The study of oxidative stress and its relation to cell death pathways also provides insights into mechanism of cellular stress. In this study we have used multiparametric assays that correlate changes in mitochondria with different cell health parameters using plate-based microcapillary cytometry on guava easyCyte platforms. Compound screening studies were performed on Jurkats (suspension) and HeLa adherent cell lines using an array of over 80 compounds to study inter-relationships between mitochondrial perturbations and different cell health parameters. Compounds were evaluated for mitochondrial depolarization and relationship to apoptosis and cell death; mitochondrial perturbations with regard to caspase levels were also evaluated; and the parallel connection between mitochondrial superoxide stress and apoptosis was also studied. Our studies identified several hit compounds, examples of which included compounds like gambogic acid, thimerosal, camtothecin, antimycin A, staurosporine, celastrol and juglone which caused significant mitochondrial perturbation and apoptosis. In addition, compounds such as antimycin A and staurosporine caused significant oxidative stress. Simplified and rapid screening assays that provide information on multiple parameters of cellular stress and apoptosis can greatly benefit the identification of hit compounds and provide a more comprehensive understanding of the mechanism of compound toxicity. Citation Format: Katherine Gillis, Julie Clor, Kimvan Tran, Asima Khan, Kamala Tyagarajan. Multiparametric approaches to evaluate mitochondrial toxicity, oxidative stress and apoptosis in drug discovery studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2013-1738

Networking the Leishmania research community for the development of novel anti-leishmanial intervention strategies

The EU-sponsored LEISHDRUG consortium uses a highly interdisciplinary approach to reveal Leishmania signaling molecules associated with virulence of the pathogenic amastigote stage. LEISHDRUG coordinates the efforts of 13 partners across eight nations, including four institutes of the Institut Pasteur international network, with the major aim to exploit the Leishmania kinome for anti-parasitic drug development. The consortium is based on three clusters with each two interactive scientific work packages that together follow the major stages of the drug development process, including identification of hit compounds and target kinases, hit-to-lead validation and lead characterization. We use innovative drug screening concepts not applied previously on parasitic systems, including visual high-content screening to discover compounds capable to kill intracellular Leishmania amastigotes without deteriorating the host cell. This phenotype-based strategy relies on fluorescent parasites and macrophages as read-outs and will allow simultaneous assessment of anti-leishmanial activity and host cell toxicity under physiological conditions. We apply a target-based strategy utilizing recombinant Leishmania protein kinases for inhibitor identification and structure-guided drug design. The identification of appropriate target kinases, with only limited homology to their mammalian counterparts relies on in silico analysis by applying novel bioinformatic tools developed by consortium members, and in vitro assays based on their phospho-transferase activity towards recombinant Leishmania phospho-proteins. The major objectives of our consortium are (i) to screen small molecule and peptide libraries for hit compounds with leishmanicidal activity using phenotype- and target-based strategies, (ii) to identify anti-parasitic lead compounds and assess their pharmacokinetic profiles using cell-culture and experimental infection models for leishmaniasis, and (iii) to initiate lead optimization by structure-based drug design.