Selection for constrained peptides that bind to a single target protein

Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards “molecular glues” for therapeutics and diagnostics.

Cyclic constrained immunoreactive peptides from crucial P. falciparum proteins: potential implications in malaria diagnostics

Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian and Latin American region. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs-failure of detection in low transmission settings, and deletion of one of the target proteins-Histidine rich protein (HRP) are evident from multiple reports; thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides-C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5,458 cyclic constrained peptides (in duplicate) against P. falciparum infected sera. Peptides (C6, A8, B7-cyclic constrained) and (G11, DSQ, NQN-corresponding linear peptides) were fairly immunoreactive towards P. falciparum-infected sera in dot-blot assay. Using indirect ELISA, cyclic constrained peptides (C6 & B7) were found to be specific to P. falciparum infected sera and further, observed to be significantly reactive towards antibodies from field-collected P. falciparum infected sera. Notably, the structural location of the epitopes defines the reactivity, observed by the preferential recognition of cyclic constrained peptides vs linear peptides and corroborated by the homology modeling analysis of selected proteins. In conclusion, the study identified three cyclic constrained immunoreactive peptides (C6, B7 & A8) from P. falciparum secretory/surface proteins and two of them (C6 & B7) were validated for their diagnostic potential with field-collected P. falciparum infected sera samples.

Rational Design of Constrained Peptides as Protein Interface Inhibitors

The lack of progress in developing targeted therapeutics directed at protein–protein complexes has been due to the absence of well-defined ligand-binding pockets and the extensive intermolecular contacts at the protein–protein interface. Our laboratory has developed approaches to dissect protein–protein complexes focusing on the superfamilies of erbB and tumor necrosis factor (TNF) receptors by the combined use of structural biology and computational biology to facilitate small molecule development. We present a perspective on the development and application of peptide inhibitors as well as immunoadhesins to cell surface receptors performed in our laboratory.