Cancer-related somatic mutations in transmembrane helices alter adenosine A1 receptor pharmacology

Overexpression of the adenosine A1 receptor (A1AR) has been detected in various cancer cell lines. However, the role of A1AR in tumor development is still unclear. Thirteen A1AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. We have investigated the pharmacology of the mutations located at the 7-transmembrane domain using a yeast system. Concentration-growth curves were obtained with the full agonist CPA and compared to the wild type hA1AR. H78L3.23 and S246T6.47 showed increased constitutive activity, while only the constitutive activity of S246T6.47 could be reduced to wild type levels by the inverse agonist DPCPX. Decreased constitutive activity was observed on five mutant receptors, among which A52V2.47 and W188C5.46 showed a diminished potency for CPA. Lastly, a complete loss of activation was observed in five mutant receptors. A selection of mutations was also investigated in a mammalian system, showing comparable effects on receptor activation as in the yeast system, except for residues pointing toward the membrane. Taken together, this study will enrich the view of the receptor structure and function of A1AR, enlightening the consequences of these mutations in cancer. Ultimately, this may provide an opportunity for precision medicine for cancer patients with pathological phenotypes involving these mutations.Medicinal Chemistr

Cancer-related somatic mutations alter adenosine A1 receptor pharmacology: a focus on mutations in the loops and C- terminus

G protein-coupled receptors (GPCRs) are known to be involved in tumor progression and metastasis. The adenosine A1 receptor (A1 AR) has been detected to be over-expressed in various cancer cell lines. However, the role of A1 AR in tumor development is not yet well characterized. A series of A1 AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. In this study, we have investigated the pharmacology of mutations located outside of the 7-transmembrane domain by using a "single-GPCR-one-G protein" yeast system. Concentration-growth curves were obtained with the full agonist CPA for 12 mutant receptors and compared to the wild-type hA1 AR. Most mutations located at the extracellular loops (EL) reduced the levels of constitutive activity of the receptor and agonist potency. For mutants at the intracellular loops (ILs) of the receptor, an increased constitutive activity was found for mutant receptor L211R5.69 , while a decreased constitutive activity and agonist response were found for mutant receptor L113F34.51 . Lastly, mutations identified on the C-terminus did not significantly influence the pharmacological function of the receptor. A selection of mutations was also investigated in a mammalian system. Overall, similar effects on receptor activation compared to the yeast system were found with mutations located at the EL, but some contradictory effects were observed for mutations located at the IL. Taken together, this study will enrich the insight of A1 AR structure and function, enlightening the consequences of these mutations in cancer. Ultimately, this may provide potential precision medicine in cancer treatment.Medicinal Chemistr

Cancer-associated mutations of the adenosine A2A receptor have diverse influences on ligand binding and receptor functions

The adenosine A(2A) receptor (A(2A)AR) is a class A G-protein-coupled receptor (GPCR). It is an immune checkpoint in the tumor micro-environment and has become an emerging target for cancer treatment. In this study, we aimed to explore the effects of cancer-patient-derived A(2A)AR mutations on ligand binding and receptor functions. The wild-type A(2A)AR and 15 mutants identified by Genomic Data Commons (GDC) in human cancers were expressed in HEK293T cells. Firstly, we found that the binding affinity for agonist NECA was decreased in six mutants but increased for the V275A mutant. Mutations A165V and A265V decreased the binding affinity for antagonist ZM241385. Secondly, we found that the potency of NECA (EC50) in an impedance-based cell-morphology assay was mostly correlated with the binding affinity for the different mutants. Moreover, S132L and H278N were found to shift the A(2A)AR towards the inactive state. Importantly, we found that ZM241385 could not inhibit the activation of V275A and P285L stimulated by NECA. Taken together, the cancer-associated mutations of A(2A)AR modulated ligand binding and receptor functions. This study provides fundamental insights into the structure-activity relationship of the A(2A)AR and provides insights for A(2A)AR-related personalized treatment in cancer.Toxicolog

Novel Cephalosporin conjugates display potent and selective inhibition of Imipenemase-type Metallo-β-Lactamases

Medicinal Chemistr

Structural Mapping of Adenosine Receptor Mutations: Ligand Binding and Signaling Mechanisms

TrendsRecent technological advances in membrane protein crystallization have resulted in a nearly exponential increase of available receptor structures. The AR family is an important example in this respect. Crystal structures of antagonist- and agonist-bound adenosine A2A receptor have recently been supplemented by a fully activated conformation in complex with a G-protein mimic, and by antagonist bound structures of the A1 receptor.SDM experiments have been essential to identify residues involved in molecular interactions between ARs and their ligands. Leveraging on recent crystal structures, this vast amount of data can now be systematically classified and interconnected with chemical and structural information of ligands and receptors.The mapping of mutational data onto crystal structures provides new understanding of molecular interactions involved in ligand recognition. Together with computational modeling, this can be used as a roadmap to create novel hypotheses and assist in the design of more systematic mutagenesis studies to answer remaining structural and functional questions.The four adenosine receptors (ARs), A1, A2A, A2B, and A3, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A2A and inactive conformations of the A1 ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family.Keywords: G protein-coupled receptor, adenosine receptor, mutagenesis, chemical modulationMedicinal Chemistr

Pan-cancer functional analysis of somatic mutations in G protein-coupled receptors

G Protein-coupled receptors (GPCRs) are the most frequently exploited drug target family, moreover they are often found mutated in cancer. Here we used a dataset of mutations found in patient samples derived from the Genomic Data Commons and compared it to the natural human variance as exemplified by data from the 1000 genomes project. We explored cancer-related mutation patterns in all GPCR classes combined and individually. While the location of the mutations across the protein domains did not differ significantly in the two datasets, a mutation enrichment in cancer patients was observed among class-specific conserved motifs in GPCRs such as the Class A "DRY" motif. A Two-Entropy Analysis confirmed the correlation between residue conservation and cancer-related mutation frequency. We subsequently created a ranking of high scoring GPCRs, using a multi-objective approach (Pareto Front Ranking). Our approach was confirmed by re-discovery of established cancer targets such as the LPA and mGlu receptor families, but also discovered novel GPCRs which had not been linked to cancer before such as the P2Y Receptor 10 (P2RY10). Overall, this study presents a list of GPCRs that are amenable to experimental follow up to elucidate their role in cancer.Medicinal Chemistr

Successive statistical and structure-based modeling to identify chemically novel kinase inhibitors

Kinases are frequently studied in the context of anticancer drugs. Their involvement in cell responses, such as proliferation, differentiation, and apoptosis, makes them interesting subjects in multitarget drug design. In this study, a workflow is presented that models the bioactivity spectra for two panels of kinases: (1) inhibition of RET, BRAF, SRC, and S6K, while avoiding inhibition of MKNK1, TTK, ERK8, PDK1, and PAK3, and (2) inhibition of AURKA, PAK1, FGFR1, and LKB1, while avoiding inhibition of PAK3, TAK1, and PIK3CA. Both statistical and structure-based models were included, which were thoroughly benchmarked and optimized. A virtual screening was performed to test the workflow for one of the main targets, RET kinase. This resulted in 5 novel and chemically dissimilar RET inhibitors with remaining RET activity of 50 value of 5.1 for the most active compound. The experimental validation of inhibitors for RET strongly indicates that the multitarget workflow is able to detect novel inhibitors for kinases, and hence, this workflow can potentially be applied in polypharmacology modeling. We conclude that this approach can identify new chemical matter for existing targets. Moreover, this workflow can easily be applied to other targets as well.Toxicolog

Development of an affinity-based probe to profile endogenous human adenosine A3 receptor expression

The adenosine A3 receptor (A3AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A3AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A3AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A3AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A3AR in pathologies.Medicinal Chemistr

Potent inhibition of nicotinamide N-Methyltransferase by alkene-linked bisubstrate mimics bearing electron deficient aromatics

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.Medicinal Chemistr

Non-Sexual Transmission of Trichomonas vaginalis in Adolescent Girls Attending School in Ndola, Zambia

OBJECTIVES: To identify risk factors for trichomoniasis among young women in Ndola, Zambia. METHOD: The study was a cross-sectional study among adolescent girls aged 13-16 years in Ndola, Zambia. Study participants were recruited from schools in selected administrative areas that represented the different socio-economic strata in town. Consenting participants were interviewed about their socio-demographic characteristics; sexual behaviour; and hygiene practices. Self-administered vaginal swabs were tested for Trichomonas vaginalis. HSV-2 antibodies were determined on serum to validate the self-reported sexual activity. RESULTS: A total of 460 girls participated in the study. The overall prevalence of trichomoniasis was 27.1%, 33.9% among girls who reported that they had ever had sex and 24.7% among virgins. In multivariate analysis the only statistically significant risk factor for trichomoniasis was inconsistent use of soap. For the virgins, none of the risk factors was significantly associated with trichomoniasis, but the association with use of soap (not always versus always) and type of toilet used (pit latrine/bush versus flush toilet) was of borderline significance. CONCLUSION: We found a high prevalence of trichomoniasis in girls in Ndola who reported that they had never had sex. We postulate that the high prevalence of trichomoniasis in virgins in Ndola is due to non-sexual transmission of trichomoniasis via shared bathing water and inconsistent use of soap