Abstract
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.
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.
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
proteasome in low micromolar concentrations. The best inhibitors, i.e.,
,
,
and
, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays.
-(cyanomethyl)acetamide
(
= 5.6 µM) and carboxaldehyde-based derivative
(
= 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters
and
irreversibly inhibited the enzyme with
values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor,
, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (
= 5.2 ± 1.9 µM,
/
= 96 ± 41 M
·s
). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.
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•Consensus docking is applied to evaluate binding of polyphenols to 3CLpro.•Ellagic acid is among strongest 3CLpro inhibitors of 19 polyphenols tested.•Ellagic acid binds into active ...site by hydrogen bonding and hydrophobic forces.•3CLpro-polyphenols interactions are confirmed by surface plasmon resonance.
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.
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.
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.
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 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.
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•Novel class of N-phenylpyrrolamide based human Topo IIα inhibitors .•Well explored chemical space and SAR.•ATP-competitive inhibition of human topoisomerase IIα.•Superior cytotoxic activities compared to marketed topoisomerase poison etoposide.
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.
•Tyrosine kinase inhibitors are drugs that they can affect the endocrine system through interaction with hormone receptors.•Antiandrogenic activity was seen for erlotinib, estrogenic activity for imatinib and antithyroid activity for ibrutinib.•Dasatinib, erlotinib, nilotinib, regorafenib and sorafenib expressed antiestrogenic activity.•Glucocorticoid activity was observed for erlotinib and ibrutinib, antiglucocorticoid activity for regorafenib and sorafenib.•Imatinib in combination with 4-hydroxytamoxifen showed concentration-dependent effects on adhesion of MCF-7 cells.
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.
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