Identification Of Novel Compounds Research Articles

Abstract 4483: New drug development opportunities for PARP1

Abstract Personalized therapies allow, not only the increase of its therapeutic efficiency, but also the reduction of the secondary effects associated to the treatment of cancer. Data obtained in our group suggest that PARP1 could play a key role in the cell cycle regulation through its interaction with E2F1 transcription factor. Considering that most oncogenic processes is associated with cell cycle deregulation, the disruption of this interaction would provide a new therapeutic target of great interest and wide spectrum of indications. Therefore, our goal was to find molecules that interfere its role in cell-cycle regulation without affecting its function on DNA repair. The identification of novel compounds disrupting the PARP1/E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP-1/E2F1 protein-protein interaction, a homologous structure of BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was utilized to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to arrive at a final subset of 83 compounds, library that were incorporated to our in vitro screening, which included both transcriptional (E2F1 activity) and survival studies. Twelve compounds were finally selected, based upon their higher capacity of inhibiting E2F1 activity.This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF. AIMS Awards Program - Project A19-513, Atomwise Inc. Citation Format: Pablo Iglesias, David Moreiras, Laura Porres-Ventin, Lara Gonzalez-Rendo, Jose A. Zumalave, Victor M. Arce, Jose A. Costoya. New drug development opportunities for PARP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4483.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4495.

A high-throughput target-based screening approach for the identification and assessment of Mycobacterium tuberculosis mycothione reductase inhibitors.

The growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Computational exploration of FOXM1 inhibitors for glioblastoma: an integrated virtual screening and molecular dynamics simulation study

In this study, a comprehensive investigation of a set of phytochemicals to identify potential inhibitors for the Forkhead box protein M1 (FOXM1) was conducted. FOXM1 is overexpressed in glioblastoma (GBM) cells and plays a crucial role in cell cycle progression, proliferation, and invasion. FOXM1 inhibitors have shown promising results in preclinical studies, and ongoing clinical trials are assessing their efficacy in GBM patients. However, there are limited studies on the identification of novel compounds against this attractive therapeutic target. To address this, the NPACT database containing 1,574 phytochemicals was used, employing a hierarchical multistep docking approach, followed by an estimation of relative binding free energy. By fixing user-defined XP-dock and MM-GBSA cut-off scores of −6.096 and −37.881 kcal/mol, the chemical space was further narrowed. Through exhaustive analysis of molecular binding interactions and various pharmacokinetics profiles, we identified four compounds, namely NPACT00002, NPACT01454, NPACT00856, and NPACT01417, as potential FOXM1 inhibitors. To assess the stability of protein-ligand binding in dynamic conditions, 100 ns Molecular dynamics (MD) simulations studies were performed. Furthermore, Molecular mechanics with generalized Born and surface area solvation (MM-GBSA) based binding free energy estimations of the entire simulation trajectories revealed a strong binding affinity of all identified compounds towards FOXM1, surpassing that of the control drug Troglitazone. Based on extensively studied multistep docking approaches, we propose that these molecules hold promise as FOXM1 inhibitors for potential therapeutic applications in GBM. However, experimental validation will be necessary to confirm their efficacy as targeted therapies. Communicated by Ramaswamy H. Sarma

De Novo Generation and Identification of Novel Compounds with Drug Efficacy Based on Machine Learning.

One of the main challenges in small molecule drug discovery is finding novel chemical compounds with desirable activity. Traditional drug development typically begins with target selection, but the correlation between targets and disease remains to be further investigated, and drugs designed based on targets may not always have the desired drug efficacy. The emergence of machine learning provides a powerful tool to overcome the challenge. Herein, a machine learning-based strategy is developed for de novo generation of novel compounds with drug efficacy termed DTLS (Deep Transfer Learning-based Strategy) by using dataset of disease-direct-related activity as input. DTLS is applied in two kinds of disease: colorectal cancer (CRC) and Alzheimer's disease (AD). In each case, novel compound is discovered and identified in in vitro and in vivo disease models. Their mechanism of actionis further explored. The experimental results reveal that DTLS can not only realize the generation and identification of novel compounds with drug efficacy but also has the advantage of identifying compounds by focusing on protein targets to facilitate the mechanism study. This work highlights the significant impact of machine learning on the design of novel compounds with drug efficacy, which provides a powerful new approach to drug discovery.

In Vitro Activities of Dithiocarbamate Derivatives against Echinococcus multilocularis Metacestode Vesicles.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the severe zoonotic disease alveolar echinococcosis. New treatment options are urgently needed. Disulfiram and dithiocarbamates were previously shown to exhibit activity against the trematode Schistosoma mansoni. As both parasites belong to the platyhelminths, here we investigated whether these compounds were also active against E. multilocularis metacestode vesicles in vitro. We used an in vitro drug-screening cascade for the identification of novel compounds against E. multilocularis metacestode vesicles with disulfiram and 51 dithiocarbamates. Five compounds showed activity against E. multilocularis metacestode vesicles after five days of drug incubation in a damage marker release assay. Structure-activity relationship analyses revealed that a S-2-hydroxy-5-nitro benzyl moiety was necessary for anti-echinococcal activity, as derivatives without this group had no effect on E. multilocularis metacestode vesicles. The five active compounds were further tested for potential cytotoxicity in mammalian cells. For two compounds with low toxicity (Schl-32.315 and Schl-33.652), IC50 values in metacestode vesicles and IC50 values in germinal layer cells were calculated. The compounds were not highly active on isolated GL cells with IC50 values of 27.0 ± 4.2 µM for Schl-32.315 and 24.7 ± 11.5 µM for Schl-33.652, respectively. Against metacestode vesicles, Schl-32.315 was not very active either with an IC50 value of 41.6 ± 3.2 µM, while Schl-33.652 showed a low IC50 of 4.3 ± 1 µM and should be further investigated in the future for its activity against alveolar echinococcosis.

Identification of novel compounds and repurposing of FDA drugs for 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme of Plasmodium falciparum to combat malaria resistance

The rise of Plasmodium falciparum resistance to Artemisinin-based combination therapies (ACTs) is a significant concern in the fight against malaria. This situation calls for the search for novel anti-malarial candidates. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) is a potential target involved in various cellular processes in P. falciparum (Pf). We screened ∼0.69 billion novel compounds from the ZINC20 library and repurposed ∼1400 FDA drugs using computational drug discovery methods against PfIspC. Following our computational pipeline, we found five novel ZINC20 compounds (Z-2, Z-3, Z-10, Z-13, and Z-14) and three FDA drugs (Aliskiren, Ceftolozane, and Ombitasvir) that showed striking docking energy (ranging from −8.405 to −10.834 kcal/mol), and strong interactions with key binding site residues (Ser269, Ser270, Ser306, Asn311, Lys312, and Met360) of PfIspC. The novel anti-malarial compounds also exhibited favorable pharmacokinetics and physicochemical properties. Furthermore, through molecular dynamics simulation, we observed the stable dynamics of PfIspC–inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. Notably, the binding free energy estimation confirmed high binding affinity (varied from −11.68 to −33.16 kcal/mol) of these compounds for PfIspC. Our findings could contribute to the ongoing efforts in combating malaria and invite experimental-lab researchers for validation.

Gambogic acid inhibits HBx-mediated hepatitis B virus replication by targeting the DTX1-Notch signaling pathway

Background & AimsCurrent antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical. MethodsInitially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo. ResultsGambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA. ConclusionsHerein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.

Deep-Sea Sponges and Corals off the Western Coast of Florida-Intracellular Mechanisms of Action of Bioactive Compounds and Technological Advances Supporting the Drug Discovery Pipeline.

The majority of natural products utilized to treat a diverse array of human conditions and diseases are derived from terrestrial sources. In recent years, marine ecosystems have proven to be a valuable resource of diverse natural products that are generated to defend and support their growth. Such marine sources offer a large opportunity for the identification of novel compounds that may guide the future development of new drugs and therapies. Using the National Oceanic and Atmospheric Administration (NOAA) portal, we explore deep-sea coral and sponge species inhabiting a segment of the U.S. Exclusive Economic Zone, specifically off the western coast of Florida. This area spans ~100,000 km2, containing coral and sponge species at sea depths up to 3000 m. Utilizing PubMed, we uncovered current knowledge on and gaps across a subset of these sessile organisms with regards to their natural products and mechanisms of altering cytoskeleton, protein trafficking, and signaling pathways. Since the exploitation of such marine organisms could disrupt the marine ecosystem leading to supply issues that would limit the quantities of bioactive compounds, we surveyed methods and technological advances that are necessary for sustaining the drug discovery pipeline including in vitro aquaculture systems and preserving our natural ecological community in the future. Collectively, our efforts establish the foundation for supporting future research on the identification of marine-based natural products and their mechanism of action to develop novel drugs and therapies for improving treatment regimens of human conditions and diseases.

Plants Used for Medicine To Fight Cancer

This article explores the potential of medicinal plants as a valuable resource in the fight against cancer. It delves into their mechanisms of action, effectiveness, safety considerations, and future prospects. Notable plants like turmeric, green tea, graviola, ginseng, and garlic are discussed for their promising anticancer properties. Research highlights their ability to hinder tumor growth by interacting with critical cellular processes. Clinical trials show positive outcomes, although challenges like standardization and interactions with conventional treatments need attention. Combining plant compounds and mainstream therapies offers exciting possibilities for better results. Integrating traditional knowledge with modern research is crucial for safe and effective treatments. Overcoming regulatory and ethical hurdles is essential for mainstream adoption. Looking forward, the discovery and development of novel plant-derived compounds hold significant promise. Personalized cancer treatment, blending genetic insights with targeted herbal approaches, represents an innovative path ahead. Collaboration between traditional practices and modern medicine is emphasized, spotlighting patient success stories. Looking ahead, the identification of novel compounds from plants and their subsequent development into pharmaceutical agents holds great potential. Personalized cancer treatment, combining genetic insights with targeted herbal interventions, is an exciting avenue for future exploration. This chapter underscores the urgency of collaborative efforts between traditional healing practices and modern medicine, spotlighting the inspiring experiences of patients who have benefited from integrating medicinal plants into their cancer care journey.

Bioprospecting of Adhatoda vasica for Identification of Novel Compounds using Chromatographic Methods and Screening for Anti-diabetic and Antioxidant Activity

This investigation column eluted fractions of leaf Adhatoda vasica of was assessed for its phytochemical screening, column chromatography, thin layer chromatographic studies, protease activity, anti-inflammatory activity, antidiabetic activity and antioxidant activity. Phytochemical screening reflects the presence of alkaloid, flavonoids, coumarins, terpenoids, steroids, emodin’s, Quinone’s. Column chromatography method was used for purification of bioactive compounds. Thin layer chromatographic study was carried out by using various solvent system of different type of polarity of n- butanol, acetic acid and acetone. TLC profiling shows pure band at 254nm and 366 nm. The strong “proteolytic activity” also pointed out in purified fraction of eluted fraction. In vitro anti-inflammatory activity was evaluated using albumin denaturation fraction 3, showing highest activity 75% followed by fraction 5 (62.73%), membrane stabilization assay fraction 6 (80.23%) followed by fraction 3 (64.65%) and proteinase inhibitory activity of fraction 5(88%) followed by fraction 7 (87.68%) at concentration 500 µg/ml. Aspirin (90.87%) was used as standard drug for the study of anti-inflammatory activity. In vitro antidiabetic activity was performed using Alfa amylase inhibition assay. Highest activity was showed in fraction 4 (79.05 %) and fraction 5 (77.05 %) at concentration 500 µg/ml. Antioxidant activity was performed by reducing power assay fraction number 2 has higher absorbance 1.04 at 500µg/ml followed by reducing power of column eluted fraction was compared with ascorbic acid as standard showing higher absorbance 0.93 at 500µg/ml.

Identification of novel compounds against Acinetobacter baumannii 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) via comprehensive structure-based computational approaches

Acinetobacter baumannii is one of the most serious opportunistic pathogens according to WHO. The difference between bacterial and mammalian fatty acid biosynthesis pathways makes FASII enzymes attractive targets in drug discovery. 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) from the FAS II pathway catalyze the condensation of malonyl ACP with acyl-ACP, and elongates the fatty acid chain by two carbons. To investigate potential inhibitors of the A. baumannii FabB, we used computational approaches including homology modeling, high-throughput virtual screening, molecular docking, molecular dynamics simulations, and MM-GBSA free energy calculations. After the high-throughput virtual screening, the resulting ligands were further screened using the QM-polarized ligand docking (QPLD) and induced fit docking (IFD) approaches. Molecular dynamics simulations were performed for 100 ns. And according to binding free energy calculations, we have identified nine compounds with the best binding affinities. Three of these compounds were selected for an additional 1 μs MD simulation to assess ligand stability. Two of them named L6 and L7 showed promised stability and affinity to the target. Here, we present novel compounds against A. baumannii FabB via structure-based computational approaches. These compounds might pave the way for the design of new lead structures and inhibitors for multidrug-resistant A. baumannii.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Drug Repurposing for the Treatment of Mycobacterium abscessus Infections

Abstract With the ever-increasing burden of antimicrobial resistance, the demand to introduce countermeasures becomes increasingly critical. The urgency of this need is intensified by the void in antibiotic discovery, with the identification of novel compounds declining with time. Of increasing concern is Mycobacterium abscessus, which displays high levels of intrinsic resistance that lead to poor success rates, even after lengthy drug regimens. Research tackling these issues is now focused on the repurposing of preexisting drugs for antimycobacterial use, facilitating the discovery of antimicrobial compounds amidst a crisis where novel antibiotics are sparse. Part of this includes the use of combination treatments, whereby coadministration of synergistic compounds can reduce dose requirements and slow the progression of antimicrobial resistance in the long term. In this review, we will introduce the current therapeutic options for M. abscessus and provide insight into why treatment is so burdensome. We will also compile the current updates within drug repurposing for this pathogen, including the use of unconventional agents such as antimalarial drugs, the repositioning of antituberculosis candidates and the repurposing of preexisting antibiotics, including the application of combination regimens. In addition, the in vitro drug screening platforms used in their discovery will be appraised, with the view of highlighting potential future perspectives that may help increase physiological relevance. This review provides a timely appraisal of the future of M. abscessus treatment.

Identification of Novel Compounds Inhibiting the Kinase Activity of the CDK5/p25 Complex via Direct Binding to p25.

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.

A multi-parametric high throughput assay for detecting beta-cell proliferation in dispersed primary islets.

Identification of novel compounds to selectively induce pancreatic beta-cell proliferation has the potential to restore functional beta-cell mass and insulin secretory demand in type 2 diabetes. The rarity of islet cell clusters (comprising of only 1% of the total pancreas mass) makes such a discovery a challenge. To address this obstacle a highthroughput, 384 well, plate-based multi-parametric imaging assay was developed to capture ex vivo primary islet proliferation, allowing positive identification of compounds that can selectively enhance islet beta-cell proliferation. The use of microscopy-based, high-content imaging technology enables acquisition of additional multi-parametric information such as proliferating populations in the islet beta and non beta-cells, insulin intensity, and cell counts, improving understanding of on and off target effects in primary tissue. The protocol requires access to a high-throughput microscopy platform for automated image acquisition of treated islet cells in assay plates. High content image analysis software is required to extract multiparametric cellular features and aid identification of therapeutically relevant small molecules and perturbants. Several putative beta-cell proliferative compounds have validated in this high throughput assay format, including the pleiotropic hormone prolactin [[1]] and the small molecule DYRK1A inhibitor harmine [[2]]. It is recommended to include one, or both, as positive controls to provide a reference for image analysis, give confidence in assay performance and capture potential assay variability during experimental runs. The protocol outlined specifically focuses on the multiparametric assessment of betacell proliferation in mouse and rat ex vivo islets and provides the methodology required for the collection of high quality cellular material. The high throughput, plate based assay can additionally be adapted to evaluate and quantify other disease relevant endpoints by high content microscopy and be applied to other downstream measurements. One of the caveats of a high-throughput, 384 microplate beta-cell proliferative assay is its limitations to facilitate human beta-cell proliferation detection, especially for weak activators. Adult human beta-cell proliferation is an extremely rare biological event and assessment experimentally can be donor dependent. In addition lower human islet beta-cell subpopulations require large numbers of cells for accurate rare event measurement.

Binding characteristics of pyrrole-scaffold hepatitis B virus capsid inhibitors and identification of novel potent compounds.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) are currently being evaluated in clinical trials as potential curative therapies for HBV. This study used in silico computational modeling to provide insights into the binding characteristics between the HBV core protein and two pyrrole-scaffold inhibitors, JNJ-6379 and GLP-26, both in the CAM-Normal (CAM-N) series. Molecular dynamics simulations showed that the pyrrole inhibitors displayed similar general binding-interaction patterns to NVR 3-778, another CAM-N, with hydrophobic interactions serving as the major driving force. However, consistent with their higher potency, the pyrrole inhibitors exhibited stronger nonpolar interactions with key residues in a solvent-accessible region as compared to NVR 3-778. This feature was facilitated by distinct hydrogen bond interactions of the pyrrole scaffold inhibitors with the residue 140 in chain B of the HBV core protein (L140B). Based on these findings, novel CAM-N compounds were designed to mimic the interaction with L140B residue while maximizing nonpolar interactions in the solvent-accessible region. Several 1H-pyrrole-2-carbonyl substituted pyrrolidine-based compounds with various hydrophobic side chains were synthesized and evaluated. Through analyses of the structure-activity and structure-druggability relations of a series of compounds, CU15 emerged as the most promising lead CAM-N compound, exhibiting sub-nanomolar potency and good pharmacokinetic profiles. Overall, the study demonstrated a practical approach to leverage computational methods for understanding key target binding features for rationale-based design, and for guiding the identification of novel compounds.

The Phytochemistry of Ganoderma Species and their Medicinal Potentials.

The Ganoderma genus is known for its diverse use as a functional food and therapeutic agent. This fungus has over 428 species, with Ganoderma lucidum being the most studied. The Ganoderma species produce several secondary metabolites and bioactive compounds like polysaccharides, phenols, and triterpenes, which are largely responsible for their therapeutic properties. Throughout this review, several extracts obtained from Ganoderma species have been studied to delve into their therapeutic characteristics and mechanisms. Such properties like immunomodulation, antiaging, antimicrobial, and anticancer activities have been demonstrated by several Ganoderma species and are supported by a large body of evidence. Although its phytochemicals play a vital role in its therapeutic properties, identifying the therapeutic potentials of fungal-secreted metabolites for human health-promoting benefits is a challenging task. Identification of novel compounds with distinct chemical scaffolds and their mechanism of action could help suppress the spread of rising pathogens. Thus, this review provides an updated and comprehensive overview of the bioactive components in different Ganoderma species and the underlying physiological mechanisms.

Identification of novel compounds using chromatographic methods and screening for protease activity and anti-inflammatory activity of Adhatoda vasica

Identification of novel compounds using chromatographic methods and screening for protease activity and anti-inflammatory activity of Adhatoda vasica

Comparative genomics with evolutionary lineage in Streptomyces bacteria reveals high biosynthetic potentials

Genome mining in silico approaches allow scientists to proficiently evaluate the genomic potency of secondary bioactive chemical producers and find new bioactive compounds in different bacteria. Streptomyces isone of the most ubiquitous bacterial genera in the environments, and well-known asprolific producers of diverse and valuable natural products (NPs) with significant biological activities. Mining and prioritizing of NP biosynthetic gene clusters (BGCs) would be the most important stage in the identification of novel compounds. Comparative genomics and genetic similarity network analysis of 62 Streptomyces public reference genomes demonstrated that individuals of these species exhibit a huge number of distinct NP BGCs, the most of which are cryptic and unconnected to any reported NPs with high phylogenetic variation among individuals. It was assumed that substantial heterogeneity across the varieties of species of Streptomyces drives outstanding biosynthetic and metabolic potential, making them plausible candidates for the identification of novel molecules.