Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling.

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems.

New treatment approaches for infections: alternatives to antibiotics and fecal microbiota transplantation.

causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option.

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.

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.

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.

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.

Inhibition of the SARS-CoV-2 3CL 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 3CL.

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.

Novel Selective IDO1 Inhibitors with Isoxazolo[5,4-]pyrimidin-4(5)-one Scaffold.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising target in immunomodulation of several pathological conditions, especially cancers. Here we present the synthesis of a series of IDO1 inhibitors with the novel isoxazolo[5,4-]pyrimidin-4(5)-one scaffold. A focused library was prepared using a 6- or 7-step synthetic procedure to allow a systematic investigation of the structure-activity relationships of the described scaffold.

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.

Second-generation 4,5,6,7-tetrahydrobenzo[]thiazoles as novel DNA gyrase inhibitors.

DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Building from our first generation of 4,5,6,7-tetrahydrobenzo[]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from and , with IC values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains.

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.

Functional diversity of small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are short non-protein-coding RNAs with a long-recognized role in tuning ribosomal and spliceosomal function by guiding ribose methylation and pseudouridylation at targeted nucleotide residues of ribosomal and small nuclear RNAs, respectively. SnoRNAs are increasingly being implicated in regulation of new types of post-transcriptional processes, for example rRNA acetylation, modulation of splicing patterns, control of mRNA abundance and translational efficiency, or they themselves are processed to shorter stable RNA species that seem to be the principal or alternative bioactive isoform. Intriguingly, some display unusual cellular localization under exogenous stimuli, or tissue-specific distribution.

Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region.

Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens.

Neuronal differentiation induces SNORD115 expression and is accompanied by post-transcriptional changes of serotonin receptor 2c mRNA.

The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing.

Alternative Affinity Ligands for Immunoglobulins.

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted.

Epitope Mapping of Mono- and Polyclonal Antibodies by Screening Phage-displayed Random Peptide Libraries.

Detailed knowledge of antigenic determinants is crucial when characterizing therapeutic and diagnostic antibodies, assessing vaccine effectiveness and developing epitope-based vaccines. Most epitope mapping approaches are labor intensive and costly. In this study, we evaluated panning of phage-displayed random peptide libraries against antibodies as a tool for cognate epitope identification.

Bacteriophages as scaffolds for bipartite display: designing swiss army knives on a nanoscale.

Bacteriophages have been exploited as cloning vectors and display vehicles for decades owing to their genetic and structural simplicity. In bipartite display setting, phage takes on the role of a handle to which two modules are attached, each endowing it with specific functionality, much like the Swiss army knife. This concept offers unprecedented potential for phage applications in nanobiotechnology.

Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor.

The many faces of small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are a class of evolutionally conserved non-coding RNAs traditionally associated with nucleotide modifications in other RNA species. Acting as guides pairing with ribosomal (rRNA) and small nuclear RNAs (snRNAs), snoRNAs direct partner enzymes to specific sites for uridine isomerization or ribose methylation, thereby influencing stability, folding and protein-interacting properties of target RNAs. In recent years, however, numerous non-canonical functions have also been ascribed to certain members of the snoRNA group, ranging from regulation of mRNA editing and/or alternative splicing to posttranscriptional gene silencing by a yet poorly understood pathway that may involve microRNA-like mechanisms.

Exploiting microRNAs for cell engineering and therapy.

MicroRNAs (miRNAs) form a large class of non-coding RNAs that function in repression of gene expression in eukaryotes. By recognizing short stretches of nucleotides within the untranslated regions of mRNAs, miRNAs recruit partner proteins to individual transcripts, leading to mRNA cleavage or hindering of translation. Bioinformatic predictions and a wealth of data from wet laboratory studies indicate that miRNAs control expression of a large proportion of protein-coding genes, implying involvement of miRNAs in regulation of most biologic processes.

Biology and applications of small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) constitute a group of non-coding RNAs principally involved in posttranscriptional modification of ubiquitously expressed ribosomal and small nuclear RNAs. However, a number of tissue-specific snoRNAs have recently been identified that apparently do not target conventional substrates and are presumed to guide processing of primary transcripts of protein-coding genes, potentially expanding the diapason of regulatory RNAs that control translation of mRNA to proteins. Here, we review biogenesis of snoRNAs and redefine their function in light of recent exciting discoveries.

Peptide phage display as a tool for drug discovery: targeting membrane receptors.

Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities.

Progress in phage display: evolution of the technique and its application.

Phage display, the presentation of (poly)peptides as fusions to capsid proteins on the surface of bacterial viruses, celebrates its 25th birthday in 2010. The technique, coupled with in vitro selection, enables rapid identification and optimization of proteins based on their structural or functional properties. In the last two decades, it has advanced tremendously and has become widely accepted by the scientific community.

Novel inhibitors of beta-ketoacyl-ACP reductase from Escherichia coli.

Bacterial beta-ketoacyl-[acyl carrier protein] (beta-ketoacyl-ACP) reductase (FabG) is a highly conserved and ubiquitously expressed enzyme of the fatty-acid biosynthetic pathway of prokaryotic organisms that catalyzes NADPH-dependent reduction of beta-ketoacyl-ACP intermediates. Therefore, FabG represents an appealing target for the development of new antimicrobial agents. A number of trans-cinnamic acid derivatives were designed and screened for inhibitory activities against FabG from Escherichia coli.