The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16180. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

The importance of Ras in drug resistance in cancer.

In this review, we analyse the impact of oncogenic Ras mutations in mediating cancer drug resistance and the progress made in the abrogation of this resistance, through pharmacological targeting. At a physiological level, Ras is implicated in many cellular proliferation and survival pathways. However, mutations within this small GTPase can be responsible for the initiation of cancer, therapeutic resistance and failure, and ultimately disease relapse. Often termed "undruggable," Ras is notoriously difficult to target directly, due to its structure and intrinsic activity. Thus, Ras-mediated drug resistance remains a considerable pharmacological problem. However, with advances in both analytical techniques and novel drug classes, the therapeutic landscape against Ras is changing. Allele-specific, direct Ras-targeting agents have reached clinical trials for the first time, indicating there may, at last, be hope of targeting such an elusive but significant protein for better more effective cancer therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Characterization of a high throughput human stem cell cardiomyocyte assay to predict drug-induced changes in clinical electrocardiogram parameters.

Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CM's) play an increasingly important role in the safety profiling of candidate drugs. For such models to have utility a clear understanding of clinical translation is required. In the present study we examined the ability of our hIPSC-CM model to predict the clinically observed effects of a diverse set of compounds on several electrocardiogram endpoints, including changes in QT and QRS intervals. To achieve this, compounds were profiled in a novel high throughput voltage-sensitive dye platform. Measurements were taken acutely (30 min) and chronically (24 h) to ensure that responses from compounds with slow onset kinetics or that affected surface ion channel expression would be captured. In addition, to avoid issues associated with changes in free drug levels due to protein binding, assays were run in serum free conditions. Changes in hIPSC-CM threshold APD90 values correlated with compound plasma exposures that produced a +10 ms change in clinical QTc (Pearson r2 = 0.80). In addition, randomForest modeling showed high predictivity in defining TdP risk (AUROC value = 0.938). Risk associated with QRS prolongation correlated with an increase in action potential rise-time (AUROC value = 0.982). The in-depth understanding of the clinical translatability of our hIPSC-CM model positions this assay to play a key role in defining cardiac risk early in drug development. Moreover, the ability to perform longer term studies enables the detection of compounds that may not be highlighted by more acute assay formats, such as inhibitors of hERG trafficking.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells.

PURPOSE: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response. EXPERIMENTAL DESIGN: A kinobead/mass spectrometry-based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings. RESULTS: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib. CONCLUSIONS: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy.

HLA Class-II‒Restricted CD8+ T Cells Contribute to the Promiscuous Immune Response in Dapsone-Hypersensitive Patients.

HLA-B∗13:01 is associated with dapsone (DDS)-induced hypersensitivity, and it has been shown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (nitroso dapsone [DDS-NO]). However, there is a need to define the importance of the HLA association in the disease pathogenesis. Thus, DDS- and DDS-NO‒specific CD8+ T-cell clones (TCCs) were generated from hypersensitive patients expressing HLA-B∗13:01 and were assessed for phenotype and function, HLA allele restriction, and killing of target cells. CD8+ TCCs were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive and several DDS-NO‒responsive TCCs expressing a variety of TCR sequences displayed HLA class-I restriction, with the drug (metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NO‒responsive CD8+ TCCs was inhibited with HLA class-II block, with DDS-NO binding to HLA-DQB1∗05:01. These TCCs were of different origin but expressed TCRs displaying the same amino acid sequences. They were activated through a hapten pathway; displayed CD45RO, CD28, PD-1, and CTLA-4 surface molecules; secreted the same panel of effector molecules as HLA class-I‒restricted TCCs; but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA molecules to activate CD8+ TCCs, with HLA class-II‒restricted CD8+ TCCs that display hybrid CD4‒CD8 features also contributing to the promiscuous immune response that develops in patients.

Pharmacological impact of FLT3 mutations on receptor activity and responsiveness to tyrosine kinase inhibitors.

Acute myelogenous leukaemia (AML) is an aggressive blood cancer characterized by the rapid proliferation of immature myeloid blast cells, resulting in a high mortality rate. The 5-year overall survival rate for AML patients is approximately 25%. Circa 35% of all patients carry a mutation in the FLT3 gene which have a poor prognosis. Targeting FLT3 receptor tyrosine kinase has become a treatment strategy in AML patients possessing FLT3 mutations. The most common mutations are internal tandem duplications (ITD) within exon 14 and a single nucleotide polymorphism (SNP) that leads to a point mutation in the D835 of the tyrosine kinase domain (TKD). Variations in the ITD sequence and the occurrence of other point mutations that lead to ligand-independent FLT3 receptor activation create difficulties in developing personalized therapeutic strategies to overcome observed mutation-driven drug resistance. Midostaurin and quizartinib are tyrosine kinase inhibitors (TKIs) with inhibitory efficacy against FLT3-ITD, but exhibit limited clinical impact. In this review, we focus on the structural aspects of the FLT3 receptor and correlate those mutations with receptor activation and the consequences for molecular and clinical responsiveness towards therapies targeting FLT3-ITD positive AML.

A functionally defined high-density NRF2 interactome reveals new conditional regulators of ARE transactivation.

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2-/-) and disease-related gain-of-function (NRF2T80K and KEAP1-/-) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease.

Apoptosis of Leukemia Cells by Ocimum basilicum Fractions Following TNF alpha Induced Activation of JNK and Caspase 3.

PURPOSE: Leukemia, one of the major cancers, affects a large proportion of people around the world. Better treatment options for leukemia are required due to a large number of side effects associated with current therapeutic regimens. In the present study, we sought to determine the pathway of triggering apoptosis of leukemic cells by Ocimum basilicum (O. basilicum) plant extract. MATERIALS/METHODS: Methanolic extract of the O. basilicum plant material was prepared. The crude extract was fractionated into several fractions through column chromatography using ethyl acetate and n-hexane as eluting solvents. Cell viability of leukemic cells was assessed via Cell titer GLO assay and apoptosis was measured through Annexin V/PI staining. Two apoptotic molecules JNK and caspases were analyzed through western blotting while pro-inflammatory cytokines TNFα, CCL2 and CXCL8 using qPCR. Fractions were characterized through LC-MS. RESULTS: The most potent with lowest IC50 values among the fractions were BF2 (2:8 n-hexane:ethyl acetate) and BF3 (3:7 n-hexane:ethyl acetate). Cytotoxicity was associated with apoptosis. Apoptosis was found caspasedependent and P-JNK activation was detected sustained. A significant increase in the level of TNF α and a decrease in the level of CXCL8 were observed in BF2 and BF3 treated cells. CONCLUSION: The fractions of O. basilicum extract were found to kill cells following JNK pathway activation. Excellent results were obtained with BF2 and BF3 probably due to predominant Epicatechin and Cinnamic acid derivatives in these fractions.

Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers.

This article updates the guidance published in 2015 for authors submitting papers to British Journal of Pharmacology (Curtis et al., 2015) and is intended to provide the rubric for peer review. Thus, it is directed towards authors, reviewers and editors. Explanations for many of the requirements were outlined previously and are not restated here. The new guidelines are intended to replace those published previously. The guidelines have been simplified for ease of understanding by authors, to make it more straightforward for peer reviewers to check compliance and to facilitate the curation of the journal's efforts to improve standards.

New Perspectives, Opportunities, and Challenges in Exploring the Human Protein Kinome.

The human protein kinome comprises 535 proteins that, with the exception of approximately 50 pseudokinases, control intracellular signaling networks by catalyzing the phosphorylation of multiple protein substrates. While a major research focus of the last 30 years has been cancer-associated Tyr and Ser/Thr kinases, over 85% of the kinome has been identified to be dysregulated in at least one disease or developmental disorder. Despite this remarkable statistic, for the majority of protein kinases and pseudokinases, there are currently no inhibitors progressing toward the clinic, and in most cases, details of their physiologic and pathologic mechanisms remain at least partially obscure. By curating and annotating data from the literature and major public databases of phosphorylation sites, kinases, and disease associations, we generate an unbiased resource that highlights areas of unmet need within the kinome. We discuss strategies and challenges associated with characterizing catalytic and noncatalytic outputs in cells, and describe successes and new frontiers that will support more comprehensive cancer-targeting and therapeutic evaluation in the future. Cancer Res; 78(1); 15-29. ©2017 AACR.

Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells.

BACKGROUND: Roughly 80% of patients with acute myeloid leukaemia have high activity of Bruton's tyrosine-kinase (BTK) in their blast cells compared with normal haemopoietic cells, rendering the cells sensitive to the oral BTK inhibitor ibrutinib in vitro. We aimed to develop the biological understanding of the BTK pathway in acute myeloid leukaemia to identify clinically relevant diagnostic information that might define a subset of patients that should respond to ibrutinib treatment. METHODS: We obtained acute myeloid leukaemia blast cells from unselected patients attending our UK hospital between Feb 19, 2010, and Jan 20, 2014. We isolated primary acute myeloid leukaemia blast cells from heparinised blood and human peripheral blood mononuclear cells to establish the activity of BTK in response to CD117 activation. Furthermore, we investigated the effects of ibrutinib on CD117-induced BTK activation, downstream signalling, adhesion to primary bone-marrow mesenchymal stromal cells, and proliferation of primary acute myeloid leukaemia blast cells. We used the Mann-Whitney U test to compare results between groups. FINDINGS: We obtained acute myeloid leukaemia blast cells from 29 patients. Ibrutinib significantly inhibited CD117-mediated proliferation of primary acute myeloid leukaemia blast cells (p=0·028). CD117 activation increased BTK activity by inducing phosphorylated BTK in patients with CD117-positive acute myeloid leukaemia. Furthermore, ibrutinib inhibited CD117-induced activity of BTK and downstream kinases at a concentration of 100 nM or more. CD117-mediated adhesion of CD117-expressing blast cells to bone-marrow stromal cells was significantly inhibited by Ibrutinib at 500 nM (p=0·028) INTERPRETATION: As first-in-man clinical trials of ibrutinib in patients with acute myeloid leukaemia commence, the data suggest not all patients will respond. Our findings show that BTK has specific pro-tumoural biological actions downstream of surface CD117 activation, which are inhibited by ibrutinib. Accordingly, we propose that patients with acute myeloid leukaemia whose blast cells express CD117 should be considered for forthcoming clinical trials of ibrutinib. FUNDING: Worldwide Cancer Research, The Big C, UK National Institutes for Health Research, the Humane Research Trust, the Department of Higher Education and Research of the Libyan Government, and Norwich Research Park.

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

TWEAKing for a fight with GVHD.

In this issue of Blood, Chopra et al provide convincing evidence that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) ligand acting through its receptor, fibroblast growth factor-inducible 14 (Fn14), is crucial to the intestinal apoptosis seen in graft-versus-host disease (GVHD) and associated mortality.

Epigenetics and pharmacology.

Recent advances in the understanding of gene regulation have shown there to be much more regulation of the genome than first thought, through epigenetic mechanisms. These epigenetic mechanisms are systems that have evolved to either switch off gene activity altogether, or fine-tune any existing genetic activation. Such systems are present in all genes and include chromatin modifications and remodelling, DNA methylation (such as CpG island methylation rates) and histone covalent modifications (e.g. acetylation, methylation), RNA interference by short interfering RNAs (siRNAs) and long non-coding RNAs (ncRNAs). These systems regulate genomic activity 'beyond' simple transcriptional factor inducer or repressor function of genes to generate mRNA. Epigenetic regulation of gene activity has been shown to be important in maintaining normal phenotypic activity of cells, as well as having a role in development and diseases such as cancer and neurodegenerative disorders such as Alzheimer's. Newer classes of drugs regulate epigenetic mechanisms to counteract disease states in humans. The reports in this issue describe some advances in epigenetic understanding that relate to human disease, and our ability to control these mechanisms by pharmacological means. Increasingly the importance of epigenetics is being uncovered - it is pharmacology that will have to keep pace.