Discovery of two non-UDP-mimic inhibitors of O-GlcNAc transferase by screening a DNA-encoded library.

Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure-activity relationship.

Boronic acid inhibitors of penicillin-binding protein 1b: serine and lysine labelling agents.

Penicillin-binding proteins (PBPs) contribute to bacterial cell wall biosynthesis and are targets of antibacterial agents. Here, we investigated PBP1b inhibition by boronic acid derivatives. Chemical starting points were identified by structure-based virtual screening and aliphatic boronic acids were selected for further investigations. Structure-activity relationship studies focusing on the branching of the boron-connecting carbon and quantum mechanical/molecular mechanical simulations showed that reaction barrier free energies are compatible with fast reversible covalent binding and small or missing reaction free energies limit the inhibitory activity of the investigated boronic acid derivatives. Therefore, covalent labelling of the lysine residue of the catalytic dyad was also investigated. Compounds with a carbonyl warhead and an appropriately positioned boronic acid moiety were shown to inhibit and covalently label PBP1b. Reversible covalent labelling of the catalytic lysine by imine formation and the stabilisation of the imine by dative N-B bond is a new strategy for PBP1b inhibition.

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones.

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS-CoV-2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in-house, cysteine-targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.

The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.

ATP-competitive inhibitors of human DNA topoisomerase IIα with improved antiproliferative activity based on N-phenylpyrrolamide scaffold.

ATP-competitive inhibitors of human DNA topoisomerase II show potential for becoming the successors of topoisomerase II poisons, the clinically successful anticancer drugs. Based on our recent screening hits, we designed, synthesized and biologically evaluated new, improved series of N-phenylpyrrolamide DNA topoisomerase II inhibitors. Six structural classes were prepared to systematically explore the chemical space of N-phenylpyrrolamide based inhibitors. The most potent inhibitor, 47d, had an IC50 value of 0.67 µM against DNA topoisomerase IIα. Compound 53b showed exceptional activity on cancer cell lines with IC50 values of 130 nM against HepG2 and 140 nM against MCF-7 cancer cell lines. The reported compounds have no structurally similarity to published structures, they are metabolically stable, have reasonable solubility and thus can serve as promising leads in the development of anticancer ATP-competitive inhibitors of human DNA topoisomerase IIα.

Fragment-Sized Thiazoles in Fragment-Based Drug Discovery Campaigns: Friend or Foe?

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.

DNA-encoded library screening on two validated enzymes of the peptidoglycan biosynthetic pathway.

Screening of DNA-encoded libraries is an emerging technology for discovering hits against protein targets. With the recent launch of the DELopen platform, a facile screening of 4.4 billion compounds is available to accelerate the drug discovery process. Here we report an affinity-based screening of the DELopen library for the first time. The screening was performed against two bacterial enzymes of the peptidoglycan biosynthetic pathway, N-acetylglucosamine-enolpyruvyl transferase (MurA) and d-alanine:d-alanine ligase (DdlB). Several binders were obtained and selected for off-DNA synthesis. Hits with confirmed inhibitory potency were deconstructed into smaller fragments. In this way, two new MurA inhibitors with antibacterial activity were obtained and are available for further optimization.

Investigating the specificity of endothelin-traps as a potential therapeutic tool for endothelin-1 related disorders.

BACKGROUND: Endothelin (ET)-traps are Fc-fusion proteins with a design based on the physiological receptors of ET-1. Previous work has shown that use of the selected ET-traps potently and significantly reduces different markers of diabetes pathology back to normal, non-disease levels. AIM: To demonstrate the selected ET-traps potently and significantly bind to ET-1. METHODS: We performed phage display experiments to test different constructs of ET-traps, and conducted bio-layer interferometry binding assays to verify that the selected ET-traps bind specifically to ET-1 and display binding affinity in the double-digit picomolar range (an average of 73.8 rM, n = 6). RESULTS: These experiments have confirmed our choice of the final ET-traps and provided proof-of-concept for the potential use of constructs as effective biologics for diseases associated with pathologically elevated ET-1. CONCLUSION: There is increased need for such therapeutics as they could help save millions of lives around the world.

Ligand Selection for Affinity Chromatography Using Phage Display.

Phage display coupled with in vitro affinity selection to mimic evolutionary principles has propelled the discovery of specific binding peptides and proteins for diverse applications, including affinity chromatography. By tailoring screening conditions, ligands with desired predefined properties, such as pH- or ion strength-responsive binding, can be identified from phage-displayed combinatorial peptide libraries. Initial hit peptides can be further optimized through directed evolution by focused mutagenesis and rescreening. Quantitative analysis of eluted binders with next-generation sequencing (NGS) assists in reducing enrichment bias and simplifies picking the most promising ligand candidate(s) through enrichment ranking. We describe, in detail, procedures of ligand selection for affinity chromatography using peptide phage display library screening, focused mutagenesis, and NGS. Furthermore, we outline the subsequent workflow for ligand characterization and affinity column construction.

Effects of tyrosine kinase inhibitors on androgen, estrogen α, glucocorticoid and thyroid receptors.

Modern anticancer therapies favor a targeted approach. Tyrosine kinase inhibitors (TKIs) are drugs that target molecular pathways involved in various types of malignancies. Although TKIs are safe and well tolerated, they remain not completely selective; e.g., endocrine-mediated adverse events have been observed with their use. In the present study, the effects of seven TKIs were determined on the activities of androgen receptor, estrogen receptor α (ERα), glucocorticoid receptor and thyroid receptor in vitro using stably transfected cell lines expressing firefly luciferase reporter gene: AR-EcoScreen, hERα-HeLa9903, MDA-kb2, and GH3.TRE-Luc cells, respectively. Antiandrogenic activity was seen for erlotinib, estrogenic activity for imatinib, antiestrogenic activity for dasatinib, erlotinib, nilotinib, regorafenib and sorafenib, glucocorticoid activity for erlotinib and ibrutinib, antiglucocorticoid activity for regorafenib and sorafenib, and antithyroid activity for ibrutinib. Additionally, synergism was seen for 1-5 µM dasatinib and 500 nM hydrocortisone combination for glucocorticoid activity in MDA-kb2 cells. The estrogenic activity of imatinib was confirmed as mediated through ERα, and interference of the TKIs with the reporter gene assays was ruled out in a cell-lysate-based firefly luciferase enzyme inhibition assay. Imatinib in combination with 4-hydroxytamoxifen showed concentration-dependent effects on the metabolic activity of ERα-expressing AN3CA, MCF-7, and SKOV3 cells, and on cell proliferation and adhesion of MCF-7 cells. These findings contribute to the understanding of the endocrine effects of TKIs, in terms of toxicity and effectiveness, and define the need to further evaluate the endocrine disrupting activities of TKIs to safeguard human and environmental health.

Inhibition of the SARS-CoV-2 3CLpro main protease by plant polyphenols.

The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CLpro. Of the polyphenols in initial in-vitro screening, quercetin, ellagic acid, curcumin, epigallocatechin gallate and resveratrol showed IC50 values of 11.8 µM to 23.4 µM. In-silico molecular dynamics simulations indicated stable interactions with the 3CLpro active site over 100 ns production runs. Moreover, surface plasmon resonance spectroscopy was used to measure the binding of polyphenols to 3CLpro in real time. Therefore, we provide evidence for inhibition of SARS-CoV-2 3CLpro by natural plant polyphenols, and suggest further research into the development of these novel 3CLpro inhibitors or biochemical probes.

Focused peptide library screening as a route to a superior affinity ligand for antibody purification.

Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.

Small and Simple, yet Sturdy: Conformationally Constrained Peptides with Remarkable Properties.

The sheer size and vast chemical space (i.e., diverse repertoire and spatial distribution of functional groups) underlie peptides' ability to engage in specific interactions with targets of various structures. However, the inherent flexibility of the peptide chain negatively affects binding affinity and metabolic stability, thereby severely limiting the use of peptides as medicines. Imposing conformational constraints to the peptide chain offers to solve these problems but typically requires laborious structure optimization. Alternatively, libraries of constrained peptides with randomized modules can be screened for specific functions. Here, we present the properties of conformationally constrained peptides and review rigidification chemistries/strategies, as well as synthetic and enzymatic methods of producing macrocyclic peptides. Furthermore, we discuss the in vitro molecular evolution methods for the development of constrained peptides with pre-defined functions. Finally, we briefly present applications of selected constrained peptides to illustrate their exceptional properties as drug candidates, molecular recognition probes, and minimalist catalysts.

Psoralen Derivatives as Inhibitors of Mycobacterium tuberculosis Proteasome.

Protein degradation is a fundamental process in all living organisms. An important part of this system is a multisubunit, barrel-shaped protease complex called the proteasome. This enzyme is directly responsible for the proteolysis of ubiquitin- or pup-tagged proteins to smaller peptides. In this study, we present a series of 92 psoralen derivatives, of which 15 displayed inhibitory potency against the Mycobacterium tuberculosis proteasome in low micromolar concentrations. The best inhibitors, i.e., 8, 11, 13 and 15, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays. N-(cyanomethyl)acetamide 8 (Ki = 5.6 µM) and carboxaldehyde-based derivative 15 (Ki = 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters 11 and 13 irreversibly inhibited the enzyme with Ki values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor, PR-957, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (Ki = 5.2 ± 1.9 µM, kinact/Ki = 96 ± 41 M-1·s-1). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.

Evolving a Peptide: Library Platforms and Diversification Strategies.

Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.