Lead compounds discovered from libraries: part 2

Many lead compounds with the potential to progress to viable drug candidates have been identified from libraries during the past two years. There are two key strategies most often employed to find leads from libraries: first, high-throughput biological screening of corporate compound collections; and second, synthesis and screening of project-directed libraries (i.e. target-based libraries). Numerous success stories, including the discovery of several clinical candidates, testify to the utility of chemical library collections as proven sources of new leads for drug development.     

Improving success rates for lead generation using affinity binding technologies

Affinity technologies have been applied at several stages of the drug discovery process, ranging from target identification and purification to the identification of preclinical candidates. The detection of ligand-macromolecule interactions in lead discovery is the best studied and most powerful of these techniques. Although affinity methods have been in widespread use for about a decade, only recently have many reports emerged on their utility. Primary affinity screens of large libraries of small molecules or fragments have begun to produce results for challenging targets. Furthermore, in secondary assays affinity methods are opening new avenues to tackle important medicinal chemistry tasks.     

Scaffold composition and biological relevance of screening libraries

The chemical scaffolds from which screening libraries are built have strong influence on the libraries' utility for screening campaigns. Here we present analysis of the scaffold composition of several types of commercially available screening collections and compare those compositions to those of drugs and drug candidates.     

High-throughput screening of biocatalytic activity: applications in drug discovery

Enzymes catalyze a diverse set of reactions that propel life's processes and hence serve as valuable therapeutic targets. High-throughput screening methods have become essential for sifting through large chemical libraries in search of drug candidates, and several sensitive and reliable analytical techniques have been specifically adapted to high-throughput measurements of biocatalytic activity. High-throughput biocatalytic assay platforms thus enable rapid screening against enzymatic targets, and have vast potential to impact various stages of the drug discovery process, including lead identification and optimization, and ADME/Tox assessment. These advances are paving the way for the adoption of high-throughput biocatalytic assays as an indispensable tool for the pharmaceutical industry.     

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Identification of inhibitors of heparin-growth factor interactions from combinatorial libraries of four-component condensation reactions

Chemical libraries based on four-component condensation (4CC) reactions of isocyanides were constructed to identify compounds capable of blocking heparin binding to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The reaction products in the synthesized libraries contain heparin mimetic functional groups such as carbohydrates, sulfonates, carboxylates, and hydroxy groups. These libraries have been screened for the inhibition of heparin binding to growth factors such as VEGF and bFGF. Single point screening at 5.0 microM of the 18,720 reaction products generated 26 candidates. The IC50S of these 26 compounds were determined using HPLC-purified products and 20 of the 26 showed significant inhibition of heparin binding to VEGF and/or bFGF. Eighteen of the 20 confirmed active compounds have a linear extended structure. Structures identified in this library revealed an initial relationship of structure and activity, thus providing direction for further investigation of this type of heparin mimetic libraries.     

Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67?% amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16?S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.     

Libraries from natural product-like scaffolds

Natural products are an attractive source of varied structures that exhibit potent biological activities, and desirable pharmacological profiles. Since the relatively recent advent of high-throughput organic synthesis in the drug discovery process, several design approaches have been applied to the construction of screening libraries. Libraries of natural-product derivatives, natural-product-like compounds prepared by total synthesis, and libraries derived from natural-products are several types that have been reported.     

Macromolecular therapeutics: emerging strategies for drug discovery in the postgenome era

The postgenome era offers a plethora of potential therapeutic targets. Many of these targets will be addressable using small organic molecules as drug candidates. However, certain aspects of cell function, particularly those that rely on protein-protein or protein-nucleic acid interactions, will be difficult to influence using small molecules. Thus, the possibility of using highly specific macromolecules as potential therapeutic agents is an intriguing concept. Recent developments in several areas of research have brought this possibility closer to fruition. Peptide and nucleic acid combinatorial libraries allow the generation of novel molecules having exquisite selectivity. Structural information and molecular modeling also contribute to the design of new macromolecules with therapeutic potential. Perhaps most importantly, approaches for delivering macromolecules into the cell interior have been developed and applied with considerable success. Thus, the therapeutic use of macromolecules, including oligonucleotides, peptides, and proteins, may be an idea whose time has come.     

A chemical approach to stem cell biology

Small molecule libraries have been used successfully to probe several biological systems. Recent work has translated these successes across to the field of stem cell biology. Stem cells hold promise for both modeling of early development as well as having therapeutic potential. Enhanced understanding of the molecular mechanisms that control stem cell fates as well as an improved ability to manipulate cell populations are required. Known mechanistic chemical compounds have been used with stem cells to accomplish these two goals. More recently, through the utilization of high fitness libraries in phenotype-based screens, several small molecules that control self-renewal and differentiation in stem cells have been identified. These small molecules provide useful chemical tools for both basic research and practical applications.     

COVID-19: Antiviral agents and enzyme inhibitors/receptor blockers in development

Novel 2019 coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) and coronavirus disease 2019 (COVID-19), the respiratory syndrome it causes, have shaken the world to its core by infecting and claiming the lives of many people since originating in December 2019 in Wuhan, China. World Health Organization and several states have declared a pandemic situation and state of emergency, respectively. As there is no treatment for COVID-19, several research institutes and pharmaceutical companies are racing to find a cure. Advances in computational approaches have allowed the screening of massive antiviral compound libraries to identify those that may potentially work against SARS-CoV-2. Antiviral agents developed in the past to combat other viruses are being repurposed. At the same time, new vaccine candidates are being developed and tested in preclinical/clinical settings. This review provides a detailed overview of select repurposed drugs, their mechanism of action, associated toxicities, and major clinical trials involving these agents.     

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host-pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed signi?cant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species.     

Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro-compounds which will serve as excellent lead candidates for further evaluation. At a 10-microM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.     

Deletion of mammalian sterile 20-like kinase 1 attenuates neuronal loss and improves locomotor function in a mouse model of spinal cord trauma

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.     

Therapeutic peptides

Novel peptide therapeutics are increasingly making their way into clinical application. Indeed, certain naturally derived peptides have been successful drugs for many years. With the advent of large biological and synthetic peptide libraries and high-throughput screening, many promising candidates could soon be added to the list of peptides under development. These advances have introduced new strategies for the administration of peptide drugs and improvements of clearance half-lives in vivo. Despite the potential obstacles that remain, peptide therapeutics are poised to play a significant role in the treatment of diseases ranging from Alzheimer's disease to cancer.     

Novel concepts for selection of catalytic activity

The selection of mutant enzymes with novel properties from libraries is emerging as a very powerful strategy for enzyme engineering. The past year has witnessed significant progress on several fronts: new and improved methods have been developed for the creation of libraries and advances have been made in screening and selection techniques. The results achieved demonstrate the enormous potential of the methods and leave questions open for further studies.