Synthetic Libraries Research Articles

Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development.

Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. KEY POINTS: • Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display. • Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library. • A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 μM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy.

Abstract 5552: Novel set of OX40 targeted antibodies and VHHs include agonists and antagonists for future development

Abstract Immunotherapy is one of the most effective modern treatments for cancer. While immunotherapy operates through a broad class of mechanisms, modulating immune checkpoints have provided some of the most effective approaches, including inhibition of PD-1/PD-L1 pathway, CTLA-4, LAG-3, OX40, Tim-3, KIR and TIGIT to reverse tumor immune escape.OX40, also known as CD134 or TNFRSF4, is a membrane protein expressed in CD4+ and CD8+ T cells, neutrophils and NK cells. In the process of T cell activation, OX40 plays a synergistic stimulating function by binding with ligand OX40L. OX40 agonist immunotherapy has been previously shown to improve the survival rate of glioblastoma in mice, prevent tumor growth in ovarian cancer, increase the prognostic significance of CD8+ cell infiltration in colorectal cancer, and eliminate the inhibitory function of Foxp3+ Treg cells that constitutively Express OX40. On the contrary, blocking OX40 axis is a promising strategy to treat T cell hyperactivation in autoimmune diseases. As recently reported in the literature, a two-armed, random, double blinded phase 2b clinical trial demonstrated that telazorlimab, an antagonist OX40 antibody developed by Ichnos Sciences, met primary efficacy endpoints in treating atopic dermatitis.In this research, we developed a set of novel antibodies to OX40 using a hybridoma and VHH synthetic library; the set of antibodies includes both agonists and antagonists.We developed one hybridoma origin mAb into a human framework. Agonist activity was equal to MOXR0916 in NF- κB reporter-Jurkat assay, dependent on coupling by proteinA/cell-bound CD32b, non-competing with OX40L, and epitope mapped to domain 2+3.We identified dozens of binders from our synthetic single domain VH library. After construction into a IgG1Fc fusion form, 4 VHHs were obtained with significant agonistic activity, dependent on coupling by proteinA/cell bound CD32b. Another 5 VHHs were also obtained with significant antagonistic activity without coupling by proteinA/cell-bound CD32b. The 4 agonistic VHHs were confirmed with binding to domain 1+2, competitive to OX40L’s binding.These VHHs constructs represent a promising set of novel biologics to modulate OX40—as agonists or antagonists—and serve as the basis for development of potential new therapies in the clinic. Citation Format: Chenguang Cai, Xiang Li, Shin-Chen Hou, Guoqian Sun, Ying Li, Ting Yang, Fengzhi Zhang, Qi Sun, Shiying Fu, Shuang Zhao, You'you Lin, Feizheng Xue. Novel set of OX40 targeted antibodies and VHHs include agonists and antagonists for future development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5552.

Synthetic libraries of immune cells displaying a diverse repertoire of chimaeric antigen receptors as a potent cancer immunotherapy.

Cancer immunotherapies rely on one or few specific tumour-associated antigens. However, the adaptive immune system relies on a large and diverse repertoire of antibodies for antigen recognition. Here we report the development and applicability of libraries of immune cells displaying diverse repertoires of chimaeric antigen receptors (CARs) that can recognize non-self antigens and display antigen-dependent clonal expansion, with the expanded population of tumour-specific effector cells leading to long-lasting antitumour responses in mouse models of epithelial tumours. The intravenous injection of synthetic libraries of murine CARs on TET2- T cells led to robust immunological memory and the recognition of mutated or evolved tumours, owing to the maintenance of CAR diversity. Off-the-shelf libraries of 106 murine or human CAR clones displayed on genetically modified human NK-92 cancer cells completely eliminated established tumours in mice with murine xenografts and patient-derived xenografts. Synthetically generated CAR libraries may aid the discovery of new CARs and the development of immunotherapies.

A Novel Synthetic Antibody Library with Complementarity-Determining Region Diversities Designed for an Improved Amplification Profile.

Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

ADAMTS4-specific MR probe to assess aortic aneurysms in vivo using synthetic peptide libraries

The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it’s potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

Azido Groups Hamper Glycan Acceptance by Carbohydrate Processing Enzymes.

Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structure of an azide compared to a hydroxyl. To quantitatively assess how well azides are accommodated, we have used glycosidases as tractable model enzyme systems reflecting what would also be expected for glycosyltransferases and other sugar binding/modifying proteins. In this vein, specificity constants have been measured for the hydrolysis of a series of azidodeoxy glucosides and N-acetylhexosaminides by a large number of glycosidases produced from expressed synthetic gene and metagenomic libraries. Azides at secondary carbons are not significantly accommodated, and thus, associated substrates are not processed, while those at primary carbons are productively recognized by only a small subset of the enzymes and often then only very poorly. Accordingly, in the absence of careful controls, results obtained with azide-modified sugars may not be representative of the situation with the natural sugar and should be interpreted with considerable caution. Azide incorporation can indeed provide a useful tool to monitor and detect glycosylation, but careful consideration should go into the selection of sites of azide substitution; such studies should not be used to quantitate glycosylation or to infer the absence of glycosylation activity.

Role of Antimicrobial Drug in the Development of Potential Therapeutics.

Population of the world run into several health-related emergencies among mankind and humans as it creates a challenge for the evolution of novel drug discoveries. One such can be the emergence of multidrug-resistant (MDR) strains in both hospital and community settings, which have been due to an inappropriate use and inadequate control of antibiotics that has led to the foremost human health concerns with a high impact on the global economy. So far, there has been application of two strategies for the development of anti-infective agents either by classical antibiotics that have been derived for their synthetic analogs with increased efficacy or screening natural compounds along with the synthetic compound libraries for the antimicrobial activities. However, need for newer treatment options for infectious diseases has led research to develop new generation of antimicrobial activity to further lessen the spread of antibiotic resistance. Currently, the principles aim to find novel mode of actions or products to target the specific sites and virulence factors in pathogens by a series of better understanding of physiology and molecular aspects of the microbial resistance, mechanism of infection process, and gene-pathogenicity relationship. The design various novel strategies tends to provide us a path for the development of various antimicrobial therapies that intends to have a broader and wider antimicrobial spectrum that helps to combat MDR strains worldwide. The development of antimicrobial peptides, metabolites derived from plants, microbes, phage-based antimicrobial agents, use of metal nanoparticles, and role of CRISPR have led to an exceptional strategies in designing and developing the next-generation antimicrobials. These novel strategies might help to combat the seriousness of the infection rates and control the health crisis system.

Prospects of measuring a metallicity trend and spread in globular clusters from low-resolution spectroscopy

Context.The metallicity spread, or the metallicity trend along the evolutionary sequence of a globular cluster, is a rich source of information to help understand the cluster physics (e.g., multiple populations) and stellar physics (e.g., atomic diffusion). Low-resolution integral-field-unit spectroscopy in the optical with the MUSE spectrograph is an attractive prospect if it can provide these diagnostics because it allows us to efficiently extract spectra of a large fraction of the cluster stars with only a few telescope pointings.Aims.We investigate the possibilities of full-spectrum fitting to derive stellar parameters and chemical abundances at low spectral resolution (R ∼ 2000).Methods.We reanalysed 1584 MUSE spectra of 1061 stars above the turn-off of NGC 6397 using FERRE and employing two different synthetic libraries.Results.We derive the equivalent iron abundance [Fe/H]efor fixed values of [α/Fe] (solar or enhanced). We find that (i) the interpolation schema and grid mesh are not critical for the precision, metallicity spread, and trend; (ii) with the two considered grids, [Fe/H]eincreases by ∼0.2 dex along the sub-giant branch, starting from the turn-off of the main sequence; (iii) restricting the wavelength range to the optical decreases the precision significantly; and (iv) the precision obtained with the synthetic libraries is lower than the precision obtained previously with empirical libraries.Conclusions.Full-spectrum fitting provides reproducible results that are robust to the choice of the reference grid of synthetic spectra and to the details of the analysis. The [Fe/H]eincrease along the sub-giant branch is in stark contrast with the nearly constant iron abundance previously found with empirical libraries. The precision of the measurements (0.05 dex on [Fe/H]e) is currently not sufficient to assess the intrinsic chemical abundance spreads, but this may change with deeper observations. Improvements of the synthetic spectra are still needed to deliver the full possibilities of full-spectrum fitting.

Analysis of human BCR-IgH from genomic DNA with UMI incorporation

Abstract Immune repertoire analysis can be performed from practically any biological sample as long as viable nucleic acid can be extracted. While using RNA as a template has its benefits, gDNA is often preferred as gDNA is often easier to obtain, especially from difficult samples. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may better reflect the quantity of cells. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. We developed a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted since the relationship of one V(D)J per cell will be maintained and will have a unique barcode added during the processing. The inclusion of UMI also assists with the elimination of PCR and sequencing error, which will lead to more confident analysis of low frequency clones and hypermutations. The primers were first tested on a BCR-IgH synthetic library. The sequencing result demonstrated >85% correlation with the known synthetic library gene expression. These results showed that the new gDNA BCR-IgH panel was sufficient at amplifying all known J gene targets. Presently, we have also applied this method to gDNA extracted from B cell enriched PBMC samples including the spike-in of a known BCR clone at various concentrations. To our knowledge, this is the first assay of its kind to incorporate UMI into BCR-IgH gDNA to capture both the BCR repertoire and true cell frequency.

A novel compound AD021 attenuates renal fibrosis by inhibiting transforming growth factor‐β1 in renal proximal tubule epithelial cells

Renal fibrogenesis is the common final pathway to all renal injuries including injury from diabetes that consequently leads to end‐stage renal disease (ESRD). Currently, there are no potential therapeutic drugs that are both safe and effective to prevent the progression of diabetic nephropathy (DN) patients and eventually ESRD. Transforming growth factor‐β1 (TGF‐β1) as the major player of tubulointerstitial fibrosis induction in diabetic kidneys. Moreover, inhibition of the TGF‐β1 pathway is one of the most effective renal fibrosis therapies in DN. Therefore, we aimed to identify the novel anti‐fibrotic compound using TGF‐β1‐induced fibrosis cells. Renal proximal tubule epithelial cells (RPTEC) were used to evaluate the anti‐fibrotic effect of the natural compounds. Our laboratory has natural and synthetic compounds libraries and some of them have been reported to have beneficial effects in several diseases, but the anti‐fibrotic effect has never been studied. The picro‐sirius red staining and spectrophotometric analysis were used for screening of 335 compounds. The active compound, AD021, effectively inhibited TGF‐β1‐ induced fibrosis in RPTEC cells with IC50 of 11 μM. Profibrotic protein expression levels including fibronectin and α‐smooth muscle actin were decreased in AD021 co‐treatment with TGF‐β1 in a concentration‐dependent manner. Collectively, our findings demonstrated that this novel anti‐fibrotic agent has potential for the development of novel renal fibrosis treatment for DN patients.

Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin - the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

Identifying Small-Molecule Inhibitors of SARS-CoV-2 RNA-Dependent RNA Polymerase by Establishing a Fluorometric Assay.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).

Puckering the Planar Landscape of Fragments: Design and Synthesis of a 3D Cyclobutane Fragment Library.

Fragment‐based drug discovery (FBDD) has a growing need for unique screening libraries. The cyclobutane moiety was identified as an underrepresented yet attractive three‐dimensional (3D) scaffold. Synthetic strategies were developed via a key 3‐azido‐cyclobutanone intermediate, giving potential access to a range of functional groups with accessible growth vectors. A focused set of 33 novel 3D cyclobutane fragments was synthesised, comprising three functionalities: secondary amines, amides, and sulfonamides. This library was designed using Principal Component Analysis (PCA) and an expanded version of the rule of three (RO3), followed by Principal Moment of Inertia (PMI) analysis to achieve both chemical diversity and high 3D character. Cis and trans ring isomers of library members were generated to maximise the shape diversity obtained, while limiting molecular complexity through avoiding enantiomers. Property analyses of the cyclobutane library indicated that it fares favourably against existing synthetic 3D fragment libraries in terms of shape and physicochemical properties.

Structural Insights into the Design of Synthetic Nanobody Libraries.

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

A Synthetic Gene Library Yields a Previously Unknown Glycoside Phosphorylase That Degrades and Assembles Poly-β-1,3-GlcNAc, Completing the Suite of β-Linked GlcNAc Polysaccharides.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP β-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosaminide linkages. The resulting poly-β-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible β-linked GlcNAc homopolysaccharides together with chitin (β-1,4) and PNAG (poly-β-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

Exploration of Chemical Biology Approaches to Facilitate the Discovery and Development of Novel Antibiotics

Approximately 2.8 million people worldwide are infected with bacteria that are deemed resistant to clinically relevant antibiotics. This accounts for 700,000 deaths every year and represents a major public health threat that has been on the rise for the past two decades. In contrast, the pace of antibiotic discovery to treat these resistant pathogens has significantly decreased. Most antibiotics are complex natural products that were isolated from soil microorganisms during the golden era of antibiotic discovery (1940s to 1960s) employing the “Waksman platform”. After the collapse of this discovery platform, other strategies and approaches emerged, including phenotype- or target-based screenings of large synthetic compound libraries. However, these methods have not resulted in the discovery and/or development of new drugs for clinical use in over 30 years. A better understanding of the structure and function of the molecular components that constitute the bacterial system is of paramount importance to design new strategies to tackle drug-resistant pathogens. Herein, we review the traditional approaches as well as novel strategies to facilitate antibiotic discovery that are chemical biology-focused. These include the design and application of chemical probes that can undergo bioorthogonal reactions, such as copper (I)-catalyzed azide-alkyne cycloadditions (CuAAC). By specifically interacting with bacterial proteins or being incorporated in the microorganism’s metabolism, chemical probes are powerful tools in drug discovery that can help uncover new drug targets and investigate the mechanisms of action and resistance of new antibacterial leads.

Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries.

This article presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules to identify B and T cell epitopes. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Screening peptide library for antibody inhibition Basic Protocol 2: Screening a peptide library to identify CD4+ Or CD8+ T cell ligands Support Protocol 1: Optimizing antigen and antibody concentrations for screening assay Support Protocol 2: Preparing a positional scanning peptide library.