Rapid and low-cost multiplex synthesis of chemokine analogs

Abstract

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size. by guest on July 4, 2020 http://www.jbc.org/ Downloaded from Figure 1. Design and evaluation of a streamlined process for rapid and inexpensive production of large panels of chemokine analogs. Until now (left panel), work exploring chemokine structure-activity relationships has been based on chemical synthesis of individual molecules. In this study (right panel), we designed and evaluated a process for the multiplex production of chemokine analogs. Black boxes, steps performed in series; red boxes, multiplex steps; blue boxes, column chromatography steps; green boxes, multiplex procedures used to replace column chromatography steps. Multiplex synthesis of chemokine analogs

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

“…Human CCL5 and reference standard [6P4]CCL5 were prepared by chemical synthesis as previously described ( 10 , 11 ). [6P4]CCL5 variants (D5A and D5K), as well as a sample of unmodified [6P4]CCL5, were prepared using a previously described multiplex chemical synthesis approach ( 49 ).…”

“…Cells were used in Ca 2+ flux experiments 24 hours later. For experiments involving [6P4]CCL5 variants, HEK ( 10 ) and CHO ( 49 ) cell clones stably expressing CCR5 (HEK-CCR5 and CHO-CCR5, respectively) were used.…”

Chemoenzymatic Semisynthesis of Proteins

“…The class of chemotactic cytokines known as chemokines have been accessed using both totally synthetic ,− and semisynthetic − ,,, protein chemistry. Typically, these proteins are only 70–80 residues and have conserved cysteine residues that are conveniently located for synthetic manipulation.…”

In‐Plate Chemical Synthesis of Isopeptide‐Linked SUMOylated Peptide Fluorescence Polarization Reagents for High‐Throughput Screening of SENP Preferences

“…To selectively SUMOylate the target lysine, we envisioned that a one‐pot NCL and desulfurization approach between a γ‐thiolysine containing peptide and a SUMO3 thioester (peptide 1) would be the most effective approach [29] . Concentration, pH and thiol catalyst are essential reaction parameters during NCL and challenging to control simultaneously in 96‐well plate format, thus a robust and optimized NCL protocol was required [30] . To add an additional challenge to this synthesis we aimed to perform the reaction under native conditions as Bouchenna et al.…”