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BACKGROUND AND AIMS As part of the renal filtration barrier, podocytes are critical to the function of kidney glomeruli. Their loss is one of the hallmarks of chronic kidney disease, including diabetic kidney disease (DKD). Indeed, many drug discovery efforts focus on preventing podocyte apoptosis triggered by diabetic stressors to delay kidney disease progression. In order to discover and characterize new therapeutic agents, phenotypic screens are a powerful tool to directly screen for the desired cellular outcome. These need to be complemented by target-centric assays to support the chemical optimization of compounds. Generally, the deconvolution and confirmation of molecular targets are considered challenging, but necessary in the follow-up of phenotype-based drug discovery programmes to allow progression towards the clinic. We have combined novel technologies like high-throughput next-generation sequencing (NGS) and Cell Painting [1] to develop an unbiased approach for deconvolution of targets from phenotypic screens. As an outcome, we identified a small molecule compound that can protect conditionally immortalized human podocytes (CIHP) from apoptosis in vitro. In a second step, we were able to identify the target responsible for this effect. METHOD Phenotypic screen: CIHP [2] were treated with high glucose and palmitate, a common DKD injury model, as the main driver for apoptosis as evaluated by caspase 3/7 activation [3]. Potentially protective agents were screened against apoptosis from a library of 50 000 small molecules. We selected a hit compound (CpdX) for subsequent target deconvolution to identify the target causing the protective effect. Target deconvolution and compound profiling: We compared the molecular signatures triggered by CpdX treatment to those of a reference panel of known, bio-annotated compounds (#L3500; Selleckchem). Dimensional reduction based on UMAP and PCA applied to transcriptomic data (NGS) as well as Cell Painting fingerprints were used to identify known compounds exhibiting a similar transcriptomic and morphological profile as the CpdX. We hypothesized that the molecular targets of these compounds also constitute likely targets for the new CpdX. RESULTS We developed a screening cascade (Figure 1A) to identify novel targets for kidney disease. A first-pass phenotypic podocyte apoptosis screen delivered a potent compound, able to rescue CIHP from a diabetic challenge in vitro (Figure 1B, C). Integrated analysis and alignment of cellular changes in transcriptomic profile and morphology following CpdX or reference compound treatment enabled us to identify the serine/threonine-protein kinase Ataxia Telangiectasia Mutated (ATM) as one of the putative targets of the orphan compound. Both KU-60019, a known inhibitor of ATM [4] (Figure 1D) and CpdX showed remarkably similar effects in NGS and Cell Painting (Figure 2A, B) and were also equally protecting the cells from apoptosis in vitro (Figure 1B). From this data, we inferred that ATM is a likely effector of CpdX treatment. CONCLUSION The initial lack of specific target candidates following primary phenotypic screens is a widespread problem in drug discovery. In the absence of a target deconvolution strategy, many potential candidates cannot be progressed easily to optimization. Here, we demonstrated that a combination of state-of-the-art technologies used to benchmark hit compounds from phenotypic screens versus known bio-annotated compound libraries represent a viable approach to perform target deconvolution of hits from phenotypic screening approaches. In this work, a phenotypic screen and subsequent deconvolution identified a novel target in combination with a novel compound, making a suitable starting point for progression into a potential kidney therapeutic agent.

BACKGROUND Conventional stratification by clinical and histopathological phenotypes only approximates the heterogeneity of chronic kidney disease (CKD) and is insufficient to drive discovery of disease-modifying therapies or predict clinical outcomes. Recent advances in molecular stratification offer a mechanistic approach to disease classification but are limited by the availability of rich patient cohort datasets [1]. The National Unified Renal Translational Research Enterprise (NURTuRE) is a unique prospective cohort study for CKD and idiopathic nephrotic syndrome that comprises bio-banked patient samples from a broad range of clinical diagnoses and kidney functional states. Access to diagnostic biopsies and biofluids enables in-depth histological and molecular analysis and offers unique opportunities for a mechanistic disease understanding. Importantly, anonymized rich clinical data are available through the UK Renal Registry, allowing for a comprehensive view on CKD heterogeneity. Here we used unsupervised molecular clustering and dimensionality reduction to integrate and visualize the complex relationship between molecular, morphological and clinical data in this large cohort. We aim to generate mechanistic disease understanding for patient-centric, integrated target and biomarker discovery that will enable the development of novel precision treatments. METHODS Diagnostic kidney biopsies were obtained from the NURTuRE biobank. Samples from 286 patients comprising 16 primary diagnoses were RNA-sequenced and passed extended control of library quality and composition. Self-organizing maps (SOM) as implemented in the OposSOM R package were used for unsupervised clustering of transcriptomes, after adjusting for batch effects and sex. We used PHATE dimensionality reduction to visualize global data structure with the aim to project clinical, morphological and molecular data onto the resulting nonlinear disease trajectories. RESULTS Unsupervised clustering of kidney transcriptomes inferred five groups with distinct molecular landscapes (F, E, C, A and AB) that were generally consistent with molecular clusters previously described for CKD.1 Correlation of metagenes revealed a highly polarized global data structure with cluster C connecting F and E with the opposing clusters A and AB. Gene set enrichment analysis suggested that the polarization results from strong opposing metabolic and immune signatures, likely reflecting tubular atrophy, immune response and infiltration (Figure 1A). Consistently, cluster identity was independent of primary diagnosis but aligned with disease progression as reflected by kidney function decline (eGFR), increasing inflammation (CRP) and age range (Figure 1B). PHATE dimensionality reduction revealed complex nonlinear relations of transcriptomes aligned with disease progression and interpreted as molecular disease trajectories partitioned by SOM clusters (Figure 1C). We are now integrating real-world data, including longitudinal kidney function and free-text histopathology reports, and are expanding the molecular analysis by proteomics and genomics. Initial exploration of histopathology and eGFR series suggests a nonrandom distribution of morphological risk factors and disease progression rates across molecular clusters and trajectories, highlighting unique opportunities for drug discovery. CONCLUSIONS Molecular stratification aligns with disease progression irrespective of clinical diagnosis, reflecting common cellular and molecular mechanisms of disease associated with kidney function decline and substantial morphological changes. Further integration of complementary datasets, including clinical time series, histopathology and multiomics, will enable mechanistic interpretation of molecular clusters and trajectories with the goal of identifying novel, targetable drivers of CKD in well-defined patient subsets.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses., While Ras is a promising target for cancer therapy, development of inhibitors targeting Ras signaling has proven challenging. Here, the authors report the discovery of Rasarfin, a small molecule from a phenotypic screen on G protein-coupled receptor (GPCR) endocytosis that acts as a dual Ras and ARF6 inhibitor.

Internalization and intracellular trafficking of hormone receptors, like receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), play pivotal roles in cell responsiveness homeostasis. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior, prevalent in cancer. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify inhibitors of receptor trafficking from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor that blocks agonist-mediated internalization of AT1R and other GPCRs, which we named Rasarfin. Rasarfin also potently inhibited agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevented cancer cell proliferation. In silico modeling and in vitro studies revealed a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a new class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.

The Transient Receptor Potential A1 (TRPA1) ion channel has evolved in animals to respond to signals from a variety of sensory stimuli. Many structural determinants of its multimodal activation have been identified to date. TRPA1 activities include responses to exogenous chemical irritants, responses to endogenous inflammatory mediators, zinc, voltage, temperature or stretch and subtle yet critical modulation by calcium ions. TRPA1 has emerged as an important target for several types of pain and inflammatory conditions because of its limited expression profile and its demonstrated roles in mediating different types of pain and sensitization of peripheral sensory afferents. Despite observed species differences in channel pharmacology, recent genetic evidence in human brings some hope that preclinical efficacy in disease models will translate to patient condition. During the past decade, various groups have investigated the development of a new class of analgesic drugs or anti-tussive agents aimed at blocking TRPA1 activity in primary sensory afferents. Several companies are advancing toward clinical proof of concept studies. This review aims to summarize key advances in the understanding of TRPA1 with regard to its roles and implications for patient conditions.

During inflammation, several Transient Receptor Potential (TRP) channels are directly or indirectly activated by inflammatory signaling molecules and microenvironmental changes including heat, oxidative conditions or low pH. In either case, specific TRP isoforms participate in chains of pro- or anti-inflammatory signaling cascades often including activation of transcription factors, protein kinases and phospholipases, which result in signal integration or amplification. In a few cases, their potentials as therapeutic targets for inflammatory conditions like pruritis, cystitis, dermatitis, asthma among other conditions are investigated pre-clinically or clinically by pioneering academic groups and industries. Significant efforts are still devoted to the understanding of the detailed physiological roles played by TRP channels during inflammation. This review intends to summarize key biological findings and reports of drug discovery activities when available, in an overview of the current status and recent developments in the field.

Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as template the 2.6 A crystal structure of rabbit sarcoplasmic reticulum Ca2+-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655]. Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.

Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCe inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCe activator exhibited the opposite effect. Disruption of PKCβ, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R(2)= 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.

TRPV1 was originally characterized as an integrator of various noxious stimuli such as capsaicin, heat, and protons. TRPV1-null mice exhibit a deficiency in sensing noxious heat stimuli, suggesting that TRPV1 is one of the main heat sensors on nociceptive primary afferent neurons and a candidate target for heat hypersensitivity in chronic pain. Several different potent and selective TRPV1 antagonists have been developed by more than 50 companies since the characterization of the receptor in 1997. A consequence of this competitive interest is the crowding of patentable chemical space, because very similar in vitro screening assays are used. To circumvent this issue and to expand our understanding of TRPV1 biology, we sought to take advantage of recent advancements in automated patch-clamp technology to design a novel screening cascade. This SAR-driving assay identified novel modulators that blocked the depolarization-induced activation of outwardly-rectifying TRPV1 currents independent of agonist stimulation, and we correlated the pharmacology to three other innovative assays for higher-throughput screening. Ultimately, we have identified a screening paradigm that would have good predictive value for future TRPV1 drug discovery projects and novel chemical space with a higher probability of gaining intellectual property coverage.

Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as template the 2.6 A crystal structure of rabbit sarcoplasmic reticulum Ca2+-ATPase [Toyoshima, C., Nakasako, M., Nomura, H.Ogawa, H. (2000) Nature 405, 647-655]. Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.

FLWIN, info:eu-repo/semantics/published, Na,K-ATPase and Related Cation Pumps: Structure, Function, and Regulatory Mechanisms