GPCR Drug Discovery & Development Insights with Dr. Terry Kenakin
Q: WHAT IS THE IMPORTANCE OF GPCRs IN DRUG DISCOVERY AND DEVELOPMENT?
A: GPCRs have long been regarded as a low-hanging fruit drug target as they are conveniently located on the cell surface and control a myriad of cell functions, but at various times over the decades, they have been the most utilized target for new therapies. An important theme in drug discovery is the realization that GPCRs are Nature’s prototypical allosteric protein linking the internal signaling machinery in the cell cytosol with the outside world (receptor compartment extracellular space). What makes GPCRs especially attractive as drug targets is their versatility. Specifically, GPCRs are pleiotropically coupled to many signaling systems in the cell and with the advent of methods to discern biased signaling, we can customize the cellular signal emerging from the activated receptor through different molecular scaffolds. In addition to drugs having efficacies of varying strength, they also yield ligands with different qualities of efficacy presented to the cell. In this regard, GPCRs, historically thought to be switches, are now known to be micro-circuits taking different signals in and yielding different signals out. Through the application of quantitative pharmacology, biologists and medicinal chemists are learning to control these signaling patterns to provide more focused drugs with fewer unwanted effects.
Q: HOW HAS THIS IMPORTANCE EVOLVED IN THE LAST DECADE?
A: Arguably, there has been a perfect storm in pharmacology and drug discovery in the past 15 years brought on from two sources: molecular dynamics and the increasing availability of new functional assays to detect the outcome of drug-receptor interactions. The appreciation of receptor proteins as dynamic systems of varying micro-conformations (ensembles) has enabled the understanding of GPCR signaling bias (the ability to dissect multiple signals from the receptor to emphasize some at the expense of others). The increased availability of functional assays has allowed us to quantify these varying signals; in early years when the response was considered a monotonic signal from the receptor to the cell, there was little opportunity to visualize the texture of signaling produced by receptor activation. Now with “more eyes to see,” we can discern subtleties of receptor signaling and observe differences in drugs that previously could not be detected.
In addition, the understanding of receptor allostery has advanced with the advent of the discovery of increasing numbers of allosteric ligands. Historically, new drug screening had been conducted in an orthosteric mode (binding) thereby understandably providing orthosteric lead molecules and few allosteric ones. With increased screening in functional systems (more suited to detect allosteric molecules) has come a concomitant increase in allosteric lead molecules. As we study these new allosteric ligands, we are seeing the versatility of allosteric drug ligands through the properties of saturation of effect and practice of probe dependence, and these ideas have opened new therapeutic vistas. So, the emphasis for small molecule drug development has shifted from copies of natural neurotransmitters and hormones and molecules that sterically block them to a flexible array of allosteric molecules capable of blocking, enhancing or otherwise modifying natural signals in drug therapy. In addition, biologic molecules are emerging onto the therapeutic horizon in the form of replacement proteins, peptides, vaccines, antibodies, and nucleotide-based therapies (gene therapy, RNAi therapy). The exciting aspect of this new class is that many biologics can do things small molecules cannot do due to an interactive conversation with the human immune system.
Q: WHAT ARE THE COMMONLY STUDIED GPCR FAMILIES BEING RESEARCHED WITHIN THE LAST DECADE?
A: Historically family A GPCRs (rhodopsin family – monoamines, neurotransmitters) were the bread and butter of receptor therapies. From there, family B (secretin family – peptide) receptors emerged, but this led to an appreciation of the difficulties encountered trying to find small drug-like molecules for them. Theoretical ideas around the requirement of multiple binding domains on these receptors (so called “affinity traps”) appear to predict that a single small molecule would be insufficient to stabilize a peptide receptor active state. A paradigm shift in the approach to peptide receptors came with the realization that small molecule allosteric ligands could yield nonpeptide therapies for these targets. In addition, advances in the formulation and delivery of peptides has greatly improved the therapeutic utilization of family B receptors. Finally, there are active research programs pursuing the other 3 families of GPCRs (glutamate, adhesion and frizzled/taste) as well, an exciting prospect as the physiological function of many of these is still to be determined. In fact, proteins classified as GPCRs through their seven transmembrane domain structure with no known function (so called “orphan receptors”) form a large uncharted area in GPCR receptor pharmacology. Studies of these utilize “reverse pharmacology” in which a ligand found for an orphan receptor is then used as a probe to uncover the physiological role of the receptor and reveal new mechanisms for future therapies.
Q: WHAT INSIGHTS DO YOU KNOW ABOUT FOR FUTURE THERAPEUTIC GPCR TARGETS?
A: The most obvious change in the field has come about with the discovery of biased signaling for GPCRs. At first thought to be a new phenomenon, it is now known simply to be standard probe dependent allostery practiced by all receptor proteins. It was detected when new functional assays became available to dissect the signaling emerging from activated receptors. The fact that biased signaling is a natural behavior of standard proteins predicts that synthetic ligands will more often than not produce signaling of a different quality than natural hormones and neurotransmitters. Gone is the belief that a synthetic analog of a natural agonist will produce the same quality of signal to a cell, but only of greater or lesser magnitude. This both opens new avenues for better therapy, but also places a burden on researchers to fully characterize the effects of synthetic molecules aimed for therapy. In addition, with the great advances in screening technologies over the past years (high throughput screening, DNA-encoded libraries, virtual screening, structure-assisted screening, fraction-based screening), the present general notion indicates it is possible to “drug” (find a ligand for) any defined target. This puts the onus on the choice of protein target as being the critical and weak link in the discovery process and target validation, and thus having the target taking the center stage in discovery programs.
Q: WHAT ROLES DO GPCRs PLAY IN VIRAL THERAPEUTICS?
A: There are basically three mechanisms GPCRs could be involved in viral function:
- The first mechanism is typified by HIV-1 and AIDs. Specifically, the HIV-1 virus utilizes a GPCR (the CCR5 chemokine receptor) directly as the portal allowing cell infection. Allosteric blockers of CCR5-HIV interaction such as maraviroc are used as a therapy in this setting.
- A second viral GPCR association is virally encoded GPCRs whereby the viral genome encodes for GPCRs that are then set free in the host to support viral replication and growth. Examples of these are various herpesvirus-encoded GPCRs and US28 of human cytomegalovirus (HCMV). Many of these GPCRs are constitutively active (spontaneously form active states that signal with no need of an agonist) and thus become pathological entities in the host. Interestingly, these pose unique therapeutic problems as only an inverse agonists can be used to quell the viral GPCR signal.
- A third association is a heightened pathology resulting from the virus targeted to GPCRs in the host; in these cases, the GPCRs are targeted for symptomatic treatment. With regard to COVID-19, the pro-inflammatory GPR4 mediating leukocyte infiltration in vascular endothelium and the C5a-C5a receptor mediating platelet hyperactivity have been implicated as targets for GPCR-based therapy in COVID-19 treatment.
Q: WHAT ARE SOME OF THE LARGEST CHALLENGES RESEARCHERS ENCOUNTER WHEN STUDYING GPCRs?
A: There are two areas of heightened importance with the changing landscape of GPCR therapeutic research:
The first is accepting the premise that screening technologies may be not be such a limiting step in the discovery chain (i.e., we can find ligands for nearly all GPCR targets). The problem area becomes which target to choose? Considering that of the 30,000 genes in the human genome, there are approximations that about 3,000 are “druggable” (small molecules will change their behavior) and another 3,000 are associated with disease. This leaves an intersection of between 800- to -1,500 viable drug targets and a great many of these are GPCRs. Currently, industry and academic programs target ~300-350 targets leaving a great many still to be explored. Incorrect choice of target minimally costs programs years of precious discovery time and resources. A second hurdle is translation of in vitro observed activity to complex systems (sometimes under the control of pathophysiological processes). The advent of biased signaling which predicts complex signaling patterns from GPCRs that may be altered by cell type and the relative stoichiometry of receptors to signaling components of the cell, means that predictions of in vivo GPCR-mediated effects are not straightforward. In this regard, the rise of phenotypic screening is leading to identification of new mechanisms of GPCR control of cell function, which may be exploited by discovery.
Q: HOW FAR ALONG HAS GPCR RESEARCH EVOLVED FROM THE TRADITIONAL IN VIVO ANIMAL MODELS TO IN VITRO CELL-BASED ASSAY?
A: The history of drug discovery has been rooted in the phenomenological observation of drug effect in whole systems (animal models, isolated organs). A major stumbling block in this process has been dissimulation between human and animal material, i.e. correspondence of GPCR activity in animal test systems to humans. Biochemical binding technology followed by cellular signaling studies bridged this gap as the genome provided human drug targets and test systems devoid of species variance. This led to a “biochemical age” of discovery where simple system pharmacology (so called “synoptic pharmacology”) was de-emphasized and recombinant systems took the lead. However, with the realization that the complete system is greater than the sum of its parts, there is now a return to whole system pharmacology. Cellular systems and label-free technology enable observation of whole system pharmacology in human tissue. Ironically, this describes nearly a full circle from whole system to whole system, but with unique differences in the systems.
Q: WHY IS IT NECESSARY TO CONSIDER MULTIPLE ASSAY TYPES TO CHARACTERIZE GPCRs?
A: GPCRs are interactive systems whose behavior is intimately associated with other proteins and molecules in the cell and extracellular space. The allosteric nature of GPCRs dictates a bidirectional flow of allosteric energy between the extracellular ligand binding site and the binding sites in the cytoplasm for signaling molecules. The absence of these partners leads to differences in observed activity. Thus, the binding of efficacious agonists to bare GPCR proteins devoid of G protein partners is hugely different from systems where those G proteins are present. This extends to many other signaling components present in the cell (i.e. β-arrestin) thereby dictating the need for the complete system to reflect pharmacological activity, i.e., functional cell systems. In addition, this question can be applied to new drug screening. Specifically, when a single probe of the receptor is used to detect ligand interaction with that receptor (i.e., radioligand), then any ligands that interact with the receptor but do not report that interaction to the screening probe, will be missed (i.e., many allosteric ligands). In contrast, a functional assay utilizes the full cadre of signaling proteins in the cell interrogating the receptor and reporting alteration of receptor conformation and function when ligands bind, i.e., this is a much broader brush to paint for detection of compound detection. It can be likened to a music box with a full complement of tuning forks vs a music box with only one tuning fork.
Q: WHAT IS THE INDUSTRY TREND TO MOVE FORWARD WITH IN VITRO ASSAYS VERSUS IN VIVO EXPERIMENTATION?
A: Clearly, in vivo drug effects are preferable since this is a view of what drugs do therapeutically. However, drug concentration is variable in vivo and real time is an issue. So it is critical to link drug concentration at the target with the observed effect. In vitro experiments obviate this dissimulation in that the concentration of drug is defined and constant and the resulting drug effect can be observed. Therefore, to determine true drug value, in vitro experiments can be invaluable, i.e., if the drug is presented to the target then this effect will occur. Any dissimulation between in vitro and in vivo results therefore may be a function of variation in pharmacokinetics; this may or may not affect evaluations of the candidate value as a drug candidate, but it is an important variable in the progression process. Thus, lack of in vivo effect can be a result of failure of pharmacodynamic or pharmacokinetic effects and this must be differentiated in drug progression studies. Determination of whether pharmacokinetics limits in vivo utility is critical to the progression of new chemical scaffolds.
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