G-protein-coupled receptors (GPCRs) are the largest and most versatile cell surface receptor family with a broad repertoire of ligands and functions. We've learned an enormous amount about discovering drugs of this receptor class since the first GPCR was cloned and expressed in 1986, such that it's now well-recognized that GPCRs are the most successful target class for approved drugs. Here we take the reader through a GPCR drug discovery journey from target to the clinic, highlighting the key learnings, best practices, challenges, trends and insights on discovering drugs that ultimately modulate GPCR function therapeutically in patients. The future of GPCR drug discovery is inspiring, with more desirable drug mechanisms and new technologies enabling the delivery of better and more successful drugs.

Whilst the advent of Immune Checkpoint Blockade has revolutionized the management of cancer, a significant proportion of patients have limited or absent response to these therapies. A key cause of this immune insensitivity is the hostile solid tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. We previously identified the acid sensing G protein coupled receptor (GPCR), GPR65, as a primary determinant of these suppressive cells. In mice, genetic deletion of Gpr65 or oral administration of small molecule GPR65 inhibitors in vivo causes a profound repolarization of immunosuppressive tumor associated macrophages, an increase in infiltrating effector cells and potent anti tumor effects in syngeneic models. In TCGA data, across all tumors, patients homozygous for a hypomorphic coding variant in GPR65 (I231L) show increased overall survival, providing compelling genetic evidence of the clinical potential of GPR65 inhibition. To further explore the translational potential of GPR65 we employed a range of techniques to define the human biology of this receptor in different contexts. At the mechanistic level, single cell RNA sequencing (scRNAseq) of human PBMCs obtained from healthy donors demonstrated a pronounced effect of low pH on the myeloid compartment, with a clear polarization of these cells toward an immunosuppressive character and modulation of GPR65 expression. In parallel, pharmacological inhibition of GPR65 in human monocyte derived macrophages exposed to low pH demonstrated that equivalent gene expression changes are primarily due to GPR65 activation. To examine the relevance of these findings to the intact acidic human TME, we performed studies in fresh primary human tumor histocultures from clear cell renal cell carcinoma (ccRCC) patients with immunohistochemically confirmed high macrophage infiltration and carbonic anhydrase 9 (CA9) expression. In these cultures, GPR65 inhibition caused a dose dependent suppression of a geneset closely overlapping with that modulated by GPR65 in primary macrophages. Furthermore, we observed a marked decrease in immune suppressive IL10 secretion with coincident elevation of specific proinflammatory chemokines. Consistent with these findings, in vivo administration of a small molecule GPR65 inhibitor elicited similar changes in human CA9 expressing RCC PDX tumors implanted in myeloid boosted CD34+ stem cell engrafted NCG mice. In summary, inhibition of GPR65 provides a unique and genetically validated approach to favorably modify the immunosupressive TME with features highly conserved between mouse and human contexts. We propose that GPR65 inhibition holds significant clinical promise, with specific evidence around ccRCC as a potential standout indication. Citation Format: Barbara Cipriani, Alastair Corbin, David Miller, Alan Naylor, Faraz Khan, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Mussa Quareshy, Anastasia Nika, Preeti Singh, Gavin Knox, Darryl Turner, Satish Sankaran, Nandini Pal Basak, Toszka Bohn, Tobia Bopp, Surya Koturan, Bo Sun, Benjamin Fairfax, Tom McCarthy, Stuart Hughes. The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 668.

Background An important facet of the hostile tumor microenvironment (TME) and a recognized driver of cancer immunosuppression is extracellular acidification which results from the glycolytic nature of rapidly proliferating cancer cells. Pathios identified GPR65, a pH-sensing G protein-coupled receptor (GPCR), as the primary sensor of this acidity in human immune cells. Activation of GPR65 is particularly pronounced in macrophages where it leads to the downregulation of key chemokines and anti-tumorigenic cytokines, and the upregulation of pro-tumorigenic wound repair and remodeling factors. In dendritic cells GPR65 signaling suppresses antigen presentation pathways, whereas in T cells and natural killer cells it leads to the suppression of chemokine receptors required for effective tumor homing. Consequently, acid sensing by GPR65 can explain tumor resistance to current immunotherapies. Validating the relevance of this target in immuno-oncology, cancer patients homozygous for the hypomorphic GPR65 variant (I231L) exhibit profoundly improved survival compared to patients with other genotypes. Methods Pathios has developed PTT-4256, a first-in-class small molecule inhibitor of GPR65. The in vitro efficacy of PTT-4256 under low pH conditions was determined by cAMP screening, gene expression and cytokine release profiling of primary immune populations. Anti-tumor activity was assessed using the MC38 and B16.F10 syngeneic mouse models, whilst immunomodulatory effects in MC38 tumor-infiltrating leukocytes were profiled by targeted RNAseq. Notably, PTT-4256 is markedly more potent at human GPR65 versus the mouse receptor. To determine its full potential in patients and to develop an accurate prediction of efficacious human exposure, we employed a range of approaches including molecular pharmacology, site-directed mutagenesis, PK/PD modelling, and genetically engineered mice. Results PTT-4256 fully counteracts the low pH-driven immunosuppressive transcriptional program in human and murine immune cells and restores the secretion of key pro-inflammatory cytokines and chemokines in macrophages. PTT-4256 displays an excellent oral PK profile across species and elicits significant monotherapy efficacy in MC38 and B16.F10 mouse models. Through molecular modelling and site-directed mutagenesis we identified three key residues that explained the difference in potency of PTT-4256 between mouse and human GPR65. Incorporating these residues in a transgenic knock-in mouse fully restored human pharmacology in vitro and provided an important translational platform. Conclusions GPR65 is a key checkpoint on tumor-infiltrating immune cells that links the chronically acidic TME to tumor-promoting immunosuppression. The GPR65 inhibitor PTT-4256 restores anti-tumor immunity and demonstrates pronounced single-agent efficacy in mice, and is therefore being advanced towards clinical development. Ethics Approval Protocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care. Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.

Background High frequencies of Tumor Associated Macrophages (TAMs) are related to poor patient prognosis. The Tumor Microenvironment (TME) is characterised by resource scarcity, toxic metabolic by-products, and low pH, together creating an immunosuppressive environment which polarises TAMs towards a pro-tumorigenic state. Methods We identified the proton-sensing G-Protein-Coupled Receptor 65 (GPR65) as a key determinant of low-pH-induced immunosuppression in human cancers, specifically via modulating TAM phenotype in response to the acidic TME. The importance of GPR65 in human cancers is highlighted by three key findings: (1) cancer patients homozygous for the hypomorphic I231L variant exhibit a pronounced survival benefit, (2) GPR65 and downstream pathway genes are highly expressed in innate immune cells from all human solid tumors when assessed by single cell RNA sequencing, and (3) low pH treatment of macrophages in vitro leads to a marked suppression of inflammatory genes and an upregulation of a tissue repair signature. Results We have identified potent and selective small-molecule antagonists of human GPR65 that inhibit the low pH-induced accumulation of cAMP in recombinant cell systems and primary human macrophages with single-digit nanomolar potencies. These compounds dose-dependently prevent the low pH-driven suppression of inflammatory cytokine and chemokine genes and counteract the upregulation of pro-tumorigenic and tissue repair genes in both human and mouse macrophages. Oral administration of our exemplar compound PTT-3213 in subcutaneous MC38 tumor-bearing mice caused gene expression changes consistent with those observed in primary macrophages in vitro, indicative of a dramatic impact on the TME. Weekly dosing of PTT-3213 significantly reduced MC38 Tumor Volume (TV) compared to vehicle (46%). This monotherapy activity was comparable to bi-weekly administration of anti-PD1, whilst combination of PTT-3213 and anti-PD-1 led to a more pronounced curtailment of TV vs vehicle-treated animals (61%). In accordance with the increased expression of chemokine genes, PTT-3213 monotherapy in MC38-bearing mice markedly elevated the frequency of tumor-infiltrating NK cells (up to 22-fold). There was also an increase in the CD8+/CD4+ T cell ratio which attained statistical significance in combination with anti-PD-1. Conclusions Taken together, we have identified GPR65 as a key innate immune checkpoint and therapeutic target in solid tumors and propose that macrophage conditioning via GPR65 inhibition may provide an efficacious strategy to counteract the immunosuppressive action of the acidic TME on TAMs in patients. Ethics Approval Protocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.

The acidic tumour microenvironment (TME) and the abundance of tumour associated macrophages (TAMs) are key features of solid tumours that drive immune suppression, support tumour growth and limit the efficacy of approved therapies. We identified the pH sensing GPCR, GPR65, as a key determinant of low pH induced immune suppression in human cancers, particularly in TAMs, based on the following observations: 1) cancer patients who are homozygous for a hypomorphic coding variant in GPR65 (I231L) show a statistically significant increase in survival and altered expression of key immune system genes compared to other genotypes; 2) single cell RNA sequencing (scRNAseq) data from multiple solid tumors show that GPR65 and downstream pathway genes are ubiquitously expressed in myeloid and other innate immune cells in human cancers; and 3) low pH acting via GPR65 profoundly alters gene expression in human macrophages in vitro, bringing about a pronounced suppression of proinflammatory cytokines and a marked upregulation of tissue repair genes. These findings identify GPR65 as a novel innate immune check point, and suggest that GPR65 inhibition could be highly beneficial in cancer. Indeed, previous work has shown that genetic ablation of the GPR65 signaling pathway in B16.F10 tumour bearing mice upregulates immunostimulatory genes in TAMs and significantly reduces tumor growth1. Pathios has identified potent and selective antagonists of human GPR65 with excellent oral bioavailability and pharmacokinetics. In line with their potencies in recombinant cell systems, lead molecules are able to inhibit low pH induced cAMP elevations in primary human macrophages with IC50 values in the single digit nM range. In macrophages subjected to acidic conditions, the inhibitory effects on cAMP are accompanied by a reduction in the expression of anti inflammatory and tissue repair genes, and the enhancement of immunostimulatory genes. In particular, GPR65 inhibition counteracts the pronounced low pH induced suppression of key Type I/II interferon (IFN) response genes and chemokines such as CXCL9, CXCL10 and CXCL11. Following oral administration in tumour bearing mice, lead compounds up-regulate the expression of anti-tumor immune response genes at systemic exposures that significantly suppress GPR65 signalling in vitro. Results from tumour growth inhibition studies in mice with our GPR65 inhibitors will be also be presented. In summary, ‘Macrophage Conditioning’ via GPR65 inhibition holds substantial promise as a novel immunoncology strategy to counteract the immunosuppressive effects of the acidic TME on TAMs, and could be deployed either as monotherapy or in combination with T cell checkpoint inhibitors or other standard of care treatments. 1Nat. Immunol. 19:1319 Citation Format: Barbara Cipriani, David Miller, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Suorav Sarkar, Zoe Smith, Rhoanne McPherson, Anastasia Nika, Preeti Singh, Toszka Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2162.

Tumor-associated macrophages (TAMs) are the major innate immune component in the microenvironment of solid tumors. These cells are highly heterogeneous and plastic but often display a pronounced immunosuppressive phenotype that supports primary tumor growth and metastasis. A recently identified determinant of the immunosuppressive properties of TAMs is the activation of the pH-sensing G protein-coupled receptor, GPR65, on these cells by the acidic microenvironment that is inherent to many advanced solid tumours1. Previous work in mouse macrophages has shown that GPR65 activation leads to an elevation of inducible cAMP early repressor (ICER), an isoform of the CREM gene, which in turn suppresses the expression of a host of proinflammatory mediators1. Consistent with a high expression of GPR65 and CREM in human myeloid cells, and particularly in human TAMs, we now show that equivalent immunosuppressive signaling is also present in human macrophages in response to low pH. Further substantiating GPR65 as an innate immune checkpoint in human cancers, we also show that subjects that are homozygous for a hypomorphic coding variant in GPR65 (I231L) have a significantly improved survival across a range of cancers compared to other genotypes. This survival advantage is maintained in patients with highly glycolytic tumors that would otherwise be predicted to respond poorly to immunotherapy, and is consistent with studies showing that genetic deletion of ICER attenuates the growth of anti-PD-1-resistant B16.F10 melanoma tumors in mice1. With a view to developing a therapeutic agent able to reverse low pH-dependent immunosuppression in TAMs, we identified potent and selective small molecule inhibitors of GPR65 from a screening campaign. These molecules were able to fully ablate the acidic pH-induced expression of ICER in human macrophages with downstream implications for pro-inflammatory cytokines known to support T cell anti-tumor responses. Additional medicinal chemistry optimization led to molecules exhibiting excellent oral bioavailability in preclinical species and systemic exposure that completely suppressed GPR65 signaling following oral dosing in mice. Profiling of these inhibitors in relevant mouse tumor models is currently ongoing. In conclusion, we have (i) demonstrated that GPR65 is a key determinant of low pH-induced immunosuppression in human macrophages and thus an important innate immune checkpoint in cancer, and (ii) identified selective small molecule inhibitors of GPR65 with potent in vivo activity. This work provides the basis for developing novel “macrophage conditioning” therapeutic agents that may have utility across a range of cancers, either as single agents or in combination with other approved immunoncology drugs. 1Nat Immunol 19:1319. Citation Format: Barbara Cipriani, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Zoe Smith, Louise Healy, John Unitt, Jessica Holien, Rhoanne McPherson, Anastasia Nika, Jessy Cartier, Tozska Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. GPR65 is a critical mediator of low pH induced immunosuppressive signalling in tumor associated macrophages: Human target validation of GPR65 as a novel innate immune checkpoint and discovery of potent, selective GPR65 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1631.

Thermal shift assays (TSAs) are among the most commonly used biophysical approaches in drug discovery in both academic and industrial settings. However, the most common interpretation of the data generated by a TSA is purely qualitative, using only the change in melting temperature (ΔTm) as a metric. This has left many questions surrounding the interpretation of the data as well as whether the TSA truly correlates with other assays. TSAs also lack theoretical descriptions of the melt behavior of proteins in the presence of multiple ligands. Here we describe a novel simplified analytical framework based on "pseudoisothermal" models as well as exact thermodynamic descriptions of protein-ligand melt behavior rooted in changes in the entropy of melting. We show how the models are broad and independently applicable, in that they can describe the behavior of any macromolecule such as a protein or DNA and demonstrate good correlations with other techniques. These models are shown to give good descriptions of assay systems containing single or multiple ligands and can determine the mechanism of interaction. The models are derived from first principles, and the theoretical justification is discussed.