Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory


G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures for nearly 100 discrete GPCRs in complex with various ligands, discovering allosteric drugs that can modulate receptor signaling remains challenging. In principle, allosteric ligands have certain advantages because they bind to pockets on the receptor that do not directly impact the orthosteric site for agonist ligands, but still tune downstream signaling pathways. But identifying and validating allosteric ligands remains difficult partly due to the dynamic nature of GPCRs in native membranes. Of the more than 3700 distinct FDA-approved drugs, there are only 19 that are allosteric, and only seven target GPCRs. Many early-stage allosteric modulators fail in the latestages of the drug development pipeline because they are designed based on static structures that fail to recapitulate biological complexity. New methodologies to probe functional relevance of allosteric lead compounds in live-cell systems early in drug discovery are needed. However, the dynamic nature of allosteric sites and low-affinities of starting compounds at concentrations amenable to cell-based screening poses a significant challenge for probing protein allosteric sites. This thesis presents a novel strategy employing genetic code expansion (GCE) and bioorthogonal chemical reactions for tethering drug fragments adjacent to allosteric sites in GPCRs to increase their potency and enable fragment-based drug screening in cellular systems. C-C chemokine receptor 5 (CCR5) was selected as a model receptor for proof-of-concept studies. First, we developed a luciferase-based reporter assay to compare and optimize side-byside the efficiency of incorporation of three noncanonical amino acids (ncAAs) at three sites on CCR5 using three distinct genetic code expansion plasmid systems. Satisfactory incorporation of each of the tested ncAAs into heterologously-expressed CCR5 was achieved. Cell-based calcium mobilization assays were carried out to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered variants using heterobifunctional compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Bioorthogonal labeling of CCR5 in live cells using inverse electron demand Diels-Alder ligation was more specific and efficient than the strain promoted azide-alkyne cycloaddition reaction assessed. GCE was then employed to site-specifically introduce one of three different reactive ncAAs in CCR5 near an allosteric binding site for the drug maraviroc (mvc). Molecular dynamics simulations were used to design heterobifunctional mvc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction with the ncAAs. We synthesized a library of these analogues and employed the bioorthogonal tethering reactions to couple the analogues to the engineered CCR5 in live cells, which were assayed using cell-based signaling assays. Tetherable low-affinity mvc fragments had higher potency for CCR5 engineered with reactive ncAAs that were adjacent to the mvc binding site. The strategy we describe to tether drug fragments to GPCRs should prove useful in probing allosteric or cryptic binding sites in fragment-based GPCR-targeted drug discovery.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Sunday, March 24, 2024

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