Fragment‐Based Phenotypic Lead Discovery: Cell‐Based Assay to Target Leishmaniasis

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment‐based phenotypic lead discovery (FPLD) combines aspects of traditional fragment‐based lead discovery (FBLD), which involves the screening of small‐molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug‐like compounds in cell‐based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low‐molecular‐weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone‐marrow‐derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets.     

SHOP: A Method For Structure‐Based Fragment and Scaffold Hopping

We present a method for fragment/scaffold substitution based on protein–ligand interactions. This concept goes beyond bioisosteric replacement, which only uses the structure of the fragment to replace as query. The methodology is validated with more than 10 biological targets relevant for drug discovery.

     

A Structure‐Based Virtual Screening Approach toward the Discovery of Histone Deacetylase Inhibitors: Identification of Promising Zinc‐Chelating Groups

The inhibitors of histone deacetylases (HDACs) have drawn a great deal of attention due to their promising potential as small‐molecule therapeutics for the treatment of cancer. By means of virtual screening with docking simulations under consideration of the effects of ligand solvation, we were able to identify six novel HDAC inhibitors with IC₅₀ values ranging from 1 to 100 μM . These newly identified inhibitors are structurally diverse and have various chelating groups for the active site zinc ion, including N‐[1,3,4]thiadiazol‐2‐yl sulfonamide, N‐thiazol‐2‐yl sulfonamide, and hydroxamic acid moieties. The former two groups are included in many drugs in current clinical use and have not yet been reported as HDAC inhibitors. Therefore, they can be considered as new inhibitor scaffolds for the development of anticancer drugs by structure–activity relationship studies to improve the inhibitory activities against HDACs. Interactions with the HDAC1 active site residues responsible for stabilizing these new inhibitors are addressed in detail.     

Fueling Open‐Source Drug Discovery: 177 Small‐Molecule Leads against Tuberculosis

With the aim of fuelling open‐source, translational, early‐stage drug discovery activities, the results of the recently completed antimycobacterial phenotypic screening campaign against Mycobacterium bovis BCG with hit confirmation in M. tuberculosis H37Rv were made publicly accessible. A set of 177 potent non‐cytotoxic H37Rv hits was identified and will be made available to maximize the potential impact of the compounds toward a chemical genetics/proteomics exercise, while at the same time providing a plethora of potential starting points for new synthetic lead‐generation activities. Two additional drug‐discovery‐relevant datasets are included: a) a drug‐like property analysis reflecting the latest lead‐like guidelines and b) an early lead‐generation package of the most promising hits within the clusters identified.     

Similarity‐Based Virtual Screening with a Bayesian Inference Network

An inference network model for molecular similarity searching: The similarity search problem is modeled using inference or evidential reasoning under uncertainty. The inference network model treats similarity searching as an evidential reasoning process in which multiple sources of evidence about compounds and reference structures are combined to estimate resemblance probabilities.

     

Fragment‐Based Lead Discovery: Screening and Optimizing Fragments for Thermolysin Inhibition

Fragment‐based drug discovery has gained a foothold in today's lead identification processes. We present the application of in silico fragment‐based screening for the discovery of novel lead compounds for the metalloendoproteinase thermolysin. We have chosen thermolysin to validate our screening approach as it is a well‐studied enzyme and serves as a model system for other proteases. A protein‐targeted virtual library was designed and screening was carried out using the program AutoDock. Two fragment hits could be identified. For one of them, the crystal structure in complex with thermolysin is presented. This compound was selected for structure‐based optimization of binding affinity and improvement of ligand efficiency, while concomitantly keeping the fragment‐like properties of the initial hit. Redesigning the zinc coordination group revealed a novel class of fragments possessing K_i values as low as 128 μM , thus they provide a good starting point for further hit evolution in a tailored lead design.     

Structure‐Based and Fragment‐Based GPCR Drug Discovery

G protein‐coupled receptors (GPCRs) are an important family of membrane proteins; historically, drug discovery in this target class has been fruitful, with many of the world’s top‐selling drugs being GPCR modulators. Until recently, the modern techniques of structure‐ and fragment‐based drug discovery had not been fully applied to GPCRs, primarily because of the instability of these proteins when isolated from their cell membrane environments. Recent advances in receptor stabilisation have facilitated major advances in GPCR structural biology over the past six years, with 21 new receptor targets successfully crystallised with one or more ligands. The dramatic increase in GPCR structural information has yielded an increased use of structure‐based methods for hit identification and progression, which are reviewed herein. Additionally, a number of fragment‐based drug discovery techniques have been validated for use with GPCRs in recent years; these approaches and their use in hit identification are reviewed.     

Inhibition of Eimeria tenella CDK‐Related Kinase 2: From Target Identification to Lead Compounds

Apicomplexan parasites encompass several human‐ and animal‐pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin‐dependent kinases (CDKs) are key molecules in cell‐cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK‐like proteins, of which one—E. tenella CDK‐related kinase 2 (EtCRK2)—has already been characterized by gene cloning and expression. 1 By using the CDK‐specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK‐like proteins as potential drug targets. An X‐ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP‐competitive inhibitors. Virtual screening and “wet‐bench” high‐throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized, leading to a set of four promising lead compounds for development as EtCRK2 inhibitors.     

Fluorine NMR‐Based Screening on Cell Membrane Extracts

The possibility of measuring the action of inhibitors of specific enzymatic reactions in intact cells, cell lysates or membrane preparations represents a major advance in the lead discovery process. Despite the relevance of assaying in physiological conditions, only a small number of biophysical techniques, often requiring complex set‐up, are applicable to these sample types. Here, we demonstrate the first application of n‐fluorine atoms for biochemical screening (n‐FABS), a homogeneous and versatile assay based on ¹⁹F NMR spectroscopy, to the detection of high‐ and low‐affinity inhibitors of a membrane enzyme in cell extracts and determination of their IC₅₀ values. Our approach can allow the discovery of novel binding fragments against targets known to be difficult to purify or where membrane‐association is required for activity. These results pave the way for future applications of the methodology to these relevant and complex biological systems.     

Fragment Screening of Soluble Epoxide Hydrolase for Lead Generation—Structure‐Based Hit Evaluation and Chemistry Exploration

Soluble epoxide hydrolase (sEH) is involved in the regulation of many biological processes by metabolizing the key bioactive lipid mediator, epoxyeicosatrienoic acids. For the development of sEH inhibitors with improved physicochemical properties, we performed both a fragment screening and a high‐throughput screening aiming at an integrated hit evaluation and lead generation. Followed by a joint dose–response analysis to confirm the hits, the identified actives were then effectively triaged by a structure‐based hit‐classification approach to three prioritized series. Two distinct scaffolds were identified as tractable starting points for potential lead chemistry work. The oxoindoline series bind at the right‐hand side of the active‐site pocket with hydrogen bonds to the protein. The 2‐phenylbenzimidazole‐4‐sulfonamide series bind at the central channel with significant induced fit, which has not been previously reported. On the basis of the encouraging initial results, we envision that a new lead series with improved properties could be generated if a vector is found that could merge the cyclohexyl functionality of the oxoindoline series with the trifluoromethyl moiety of the 2‐phenylbenzimidazole‐4‐sulfonamide series.     

Toward Docking‐Based Virtual Screening for Discovering Antitubulin Agents by Targeting Taxane and Colchicine Binding Sites

Docking‐based virtual screening : Flexible docking, scoring, and virtual screening of ligand databases are on the way to fulfilling the promise. Docking‐based virtual screening that targets taxane and colchicine binding sites will certainly provide new antitubulin agents.

     

MSN‐on‐a‐Chip: Cell‐Based Screenings Made Possible on a Small‐Molecule Microarray of Native Natural Products

Standard small‐molecule microarrays (SMMs) are not well‐suited for cell‐based screening assays. Of the few attempts made thus far to render SMMs cell‐compatible, all encountered major limitations. Here we report the first mesoporous silica nanoparticle (MSN)‐on‐a‐chip platform capable of allowing high‐throughput cell‐based screening to be conducted on SMMs. By making use of a glass surface on which hundreds of MSNs, each encapsulated with a different native natural product, were immobilized in spatially defined manner, followed by on‐chip mammalian cell growth and on‐demand compound release, high‐content screening was successfully carried out with readily available phenotypic detection methods. By combining this new MSN‐on‐a‐chip system with small interfering RNA technology for the first time, we discovered that (+)‐usniacin possesses synergistic inhibitory properties similar to those of olaparib (an FDA‐approved drug) in BRCA1‐knockdown cancer cells.     

A Virtual Screening Study of the 18 kDa Translocator Protein using Pharmacophore Models Combined with 3D‐QSAR Studies

In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D‐QSAR studies. This hypothesis was used in a ligand‐based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 μM , and among them two compounds exhibited sub‐micromolar K_i values. These results validated our applied methodologies, and the two compounds with sub‐micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.     

Three‐Dimensional Database Mining Identifies a Unique Chemotype that Unites Structurally Diverse Botulinum Neurotoxin Serotype A Inhibitors in a Three‐Zone Pharmacophore

A search query consisting of two aromatic centers and two cationic centers was defined based on previously identified small molecule inhibitors of the botulinum neurotoxin serotype A light chain (BoNT/A LC) and used to mine the National Cancer Institute Open Repository. Ten small molecule hits were identified, and upon testing, three demonstrated inhibitory activity. Of these, one was structurally unique, possessing a rigid diazachrysene scaffold. The steric limitations of the diazachrysene imposed a separation between the overlaps of previously identified inhibitors, revealing an extended binding mode. As a result, the pharmacophore for BoNT/A LC inhibition has been modified to encompass three zones. To demonstrate the utility of this model, a novel three‐zone inhibitor was mined and its activity was confirmed.     

Sugar‐Based Arylsulfonamide Carboxylates as Selective and Water‐Soluble Matrix Metalloproteinase‐12 Inhibitors

Matrix metalloproteinase‐12 (MMP‐12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a β‐N‐acetyl‐d ‐glucosamine moiety, were designed and synthesized as MMP‐12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP‐12 inhibitor with improved water solubility, compound 3 [(R)‐2‐(N‐(2‐(3‐(2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl)thioureido)ethyl)biphenyl‐4‐ylsulfonamido)‐3‐methylbutanoic acid], was identified.     

Small‐Molecule APOBEC3G DNA Cytosine Deaminase Inhibitors Based on a 4‐Amino‐1,2,4‐triazole‐3‐thiol Scaffold

APOBEC3G (A3G) is a single‐stranded DNA cytosine deaminase that functions in innate immunity against retroviruses and retrotransposons. Although A3G can potently restrict Vif‐deficient HIV‐1 replication by catalyzing excessive levels of G→A hypermutation, sublethal levels of A3G‐catalyzed mutation may contribute to the high level of HIV‐1 fitness and its incurable prognosis. To chemically modulate A3G catalytic activity with the goal of decreasing the HIV‐1 genomic mutation rate, we synthesized and biochemically evaluated a class of 4‐amino‐1,2,4‐triazole‐3‐thiol small‐molecule inhibitors identified by high‐throughput screening. This class of compounds exhibits low‐micromolar (3.9–8.2 μM ) inhibitory potency and remarkable specificity for A3G versus the related cytosine deaminase, APOBEC3A. Chemical modification of inhibitors, A3G mutational screening, and thiol reactivity studies implicate C321, a residue proximal to the active site, as the critical A3G target for this class of molecules.