De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G
}, volume={1669}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2022.462941}, abstractNote={Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Xiao, Xingqing and Kilgore, Ryan and Sarma, Sudeep and Chu, Wenning and Menegatti, Stefano and Hall, Carol K.}, year={2022}, month={Apr} } @article{sarma_herrera_xiao_hudalla_hall_2022, title={Computational Design and Experimental Validation of ACE2-Derived Peptides as SARS-CoV-2 Receptor Binding Domain Inhibitors}, volume={126}, ISSN={["1520-5207"]}, DOI={10.1021/acs.jpcb.2c039188129J}, number={41}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Sarma, Sudeep and Herrera, Stephanie M. and Xiao, Xingqing and Hudalla, Gregory A. and Hall, Carol K.}, year={2022}, month={Oct}, pages={8129–8139} } @article{sarma_herrera_xiao_hudalla_hall_2022, title={Computational Design and Experimental Validation of ACE2-Derived Peptides as SARS-CoV-2 Receptor Binding Domain Inhibitors}, volume={10}, ISSN={["1520-5207"]}, DOI={10.1021/acs.jpcb.2c03918}, abstractNote={The COVID-19 pandemic has caused significant social and economic disruption across the globe. Cellular entry of SARS-CoV-2 into the human body is mediated via binding of the Receptor Binding Domain (RBD) on the viral Spike protein (SARS-CoV-2 RBD) to Angiotensin-Converting Enzyme 2 (ACE2) expressed on host cells. Molecules that can disrupt ACE2:RBD interactions are attractive therapeutic candidates to prevent virus entry into human cells. A computational strategy that combines our Peptide Binding Design (PepBD) algorithm with atomistic molecular dynamics simulations was used to design new inhibitory peptide candidates via sequence iteration starting with a 23-mer peptide, referred to as SBP1. SBP1 is derived from a region of the ACE2 Peptidase Domain α1 helix that binds to the SARS-CoV-2 RBD of the initial Wuhan-Hu-1 strain. Three peptides demonstrated a solution-phase RBD-binding dissociation constant in the micromolar range during tryptophan fluorescence quenching experiments, one peptide did not bind, and one was insoluble at micromolar concentrations. However, in competitive ELISA assays, none of these peptides could outcompete ACE2 binding to SARS-CoV-2-RBD up to concentrations of 50 μM, similar to the parent SBP1 peptide which also failed to outcompete ACE2:RBD binding. Molecular dynamics simulations suggest that P4 would have a good binding affinity for the RBD domain of Beta-B.1.351, Gamma-P.1, Kappa-B.1.617.1, Delta-B.1.617.2, and Omicron-B.1.1.529 variants, but not the Alpha variant. Consistent with this, P4 bound Kappa-B.1.617.1 and Delta-B.1.617.2 RBD with micromolar affinity in tryptophan fluorescence quenching experiments. Collectively, these data show that while relatively short unstructured peptides can bind to SARS-CoV-2 RBD with moderate affinity, they are incapable of outcompeting the strong interactions between RBD and ACE2.}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Sarma, Sudeep and Herrera, Stephanie M. and Xiao, Xingqing and Hudalla, Gregory A. and Hall, Carol K.}, year={2022}, month={Oct} } @article{xiao_robang_sarma_le_helmicki_lambert_guerrero-ferreira_arboleda-echavarria_paravastu_hall_2022, title={Sequence patterns and signatures: Computational and experimental discovery of amyloid-forming peptides}, volume={1}, ISSN={["2752-6542"]}, DOI={10.1093/pnasnexus/pgac263}, abstractNote={Screening amino acid sequence space via experiments to discover peptides that self-assemble into amyloid fibrils is challenging. We have developed a computational peptide assembly design (PepAD) algorithm that enables the discovery of amyloid-forming peptides. Discontinuous molecular dynamics (DMD) simulation with the PRIME20 force field combined with the FoldAmyloid tool is used to examine the fibrilization kinetics of PepAD-generated peptides. PepAD screening of ∼10,000 7-mer peptides resulted in twelve top-scoring peptides with two distinct hydration properties. Our studies revealed that eight of the twelve in silico discovered peptides spontaneously form amyloid fibrils in the DMD simulations and that all eight have at least five residues that the FoldAmyloid tool classifies as being aggregation-prone. Based on these observations, we re-examined the PepAD-generated peptides in the sequence pool returned by PepAD and extracted five sequence patterns as well as associated sequence signatures for the 7-mer amyloid-forming peptides. Experimental results from Fourier transform infrared spectroscopy (FTIR), thioflavin T (ThT) fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) indicate that all the peptides predicted to assemble in silico assemble into antiparallel β-sheet nanofibers in a concentration-dependent manner. This is the first attempt to use a computational approach to search for amyloid-forming peptides based on customized settings. Our efforts facilitate the identification of β-sheet-based self-assembling peptides, and contribute insights towards answering a fundamental scientific question: "What does it take, sequence-wise, for a peptide to self-assemble?".}, number={5}, journal={PNAS NEXUS}, author={Xiao, Xingqing and Robang, Alicia S. and Sarma, Sudeep and Le, Justin V. and Helmicki, Michael E. and Lambert, Matthew J. and Guerrero-Ferreira, Ricardo and Arboleda-Echavarria, Johana and Paravastu, Anant K. and Hall, Carol K.}, year={2022}, month={Nov} } @article{xiao_sarma_menegatti_crook_magness_hall_2021, title={In Silico Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Clostridium difficile Toxin A}, volume={17}, ISSN={["1554-8937"]}, url={https://doi.org/10.1021/acschembio.1c00743}, DOI={10.1021/acschembio.1c00743}, abstractNote={Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.}, number={1}, journal={ACS CHEMICAL BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Xiao, Xingqing and Sarma, Sudeep and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2021}, month={Dec} }