Structural and functional characterization of G protein-coupled receptors with deep mutational scanning.

The >800 human G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

Targeting CXCR4 in AML and ALL.

The interaction of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts with the bone marrow microenvironment regulates self-renewal, growth signaling, as well as chemotherapy resistance. The chemokine receptor, CXC receptor 4 (CXCR4), with its ligand chemokine ligand 12 (CXCL12), plays a key role in the survival and migration of normal and malignant stem cells to the bone marrow. High expression of CXCR4 on AML and ALL blasts has been shown to be a predictor of poor prognosis for these diseases. Several small molecule inhibitors, short peptides, antibodies, and antibody drug conjugates have been developed for the purposes of more effective targeting and killing of malignant cells expressing CXCR4. In this review we will discuss recent results and strategies in targeting CXCR4 with these agents in patients with AML or ALL.

Targeting VLA4 integrin and CXCR2 mobilizes serially repopulating hematopoietic stem cells.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.

A Scalable, Multiplexed Assay for Decoding GPCR-Ligand Interactions with RNA Sequencing.

G protein-coupled receptors (GPCRs) are central to how mammalian cells sense and respond to chemicals. Mammalian olfactory receptors (ORs), the largest family of GPCRs, mediate the sense of smell through activation by small molecules, though for most bonafide ligands, they have not been identified. Here, we introduce a platform to screen large chemical panels against multiplexed GPCR libraries using next-generation sequencing of barcoded genetic reporters in stably engineered human cell lines. We mapped 39 mammalian ORs against 181 odorants and identified 79 interactions that have not been reported to our knowledge, including ligands for 15 previously orphaned receptors. This multiplexed receptor assay allows the cost-effective mapping of large chemical libraries to receptor repertoires at scale.