Exploring the use of intracellular and extracellular allosteric modulators to understand GPCR signaling

Drug development was historically started by targeting protein active sites as means to pharmacologically modulate the functional properties of the target. However, with high attrition rates, pharmacologists and medicinal chemists must begin thinking outside the box more earnestly when designing new drugs. Such thinking has created an impetus toward the discovery of “biased” or “allosteric” modulators to fine-tune activity, selecting for a desired therapeutic profile. This has become a sought-after approach for the therapeutic targeting of G protein-coupled receptors (GPCRs). Structure-based studies have greatly increased our mechanistic understanding of GPCR activation in large part due to the use of single domain antibodies (or nanobodies). Even if nanobodies were initially developed to assist in receptor stabilization for structure determination of GPCRs, they have now proven to be more than simple chaperones for crystallization. Nanobodies have helped elucidate key features of GPCR biological responses, as they also behave are allosteric regulators of GPCR pharmacology. For many years, optical biosensors relying on resonance energy transfer have conventionally been used to quantify allosteric regulation. Yet, the transformation of nanobodies into biosensors, sensing distinct receptor conformation or activation states, has generated unprecedented knowledge of GPCR biology and signal transduction. Further, peptidomimetic ligands or pepducins generated from the primary and secondary structures of GPCRs have also been used as key allosteric tools to understand and drive GPCR signaling. In this review, we discuss how intracellular allosteric modulators, such as intracellular ions, and tool compounds, such as pepducins and nanobodies, have informed our understanding of G protein-coupled receptors from structural studies, to the generation of receptor conformational and signaling signatures.