Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics

Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening

There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).National Cancer Institute https://doi.org/10.13039/100000054Stony Brook Cancer CenterPeer Reviewe

FLOW INJECTION MONITORING OF ENZYME REACTIONS ON SOLID SURFACES

Optosensing flow injection analysis may be used to monitor enzyme reactionson various types of solid surfaces, e.g., cellulose, Teflon or polypropylene. The surfaces may bein the form ofa pad, a membraneorparticles (in a column). While the enzyme maybe in solution, advantages accrueif it is attached to the solid surface. Traditional enzyme immobilization on surfaces involves chemical bonding reactions, but enzymes may be directly adsorbed onto certain hydrophobic surfaces. The principles of optosensing measurements are presented andapplicationsto cellulose pads and gas barrier membranes for enzyme measurements are reviewed. Recent novel methodsfor derivatizing enzymes for direct adsorption onto fluorocarbon membranesare presented. A new gas sensing (gas gap) membrane is described in which the membraneacts as the actual sensor and notjust a barrier, suitable for optosensing of monitoring enzyme reactions. Preliminary studies of the direct adsorption of urease enzyme onto the membrane are presented. Silica based C-18 particles in a microcolumn are used to adsorb an indicator and native urease enzymé. Optosensing measurementsof urea by measuring transmittance through the particles are presented

Chemical validation and optimization of pharmacoperones targeting vasopressin type 2 receptor mutant

A series of compounds formerly identified by high-throughput screening was studied for their ability to serve as pharmacoperones for the vasopressin type 2 receptor (V2R) mutant L83Q, which is known to cause nephrogenic diabetes insipidus (NDI). Three compounds were particularly effective in rerouting the mutant receptor in a concentration-dependent manner, were neither agonists nor antagonists, and displayed low cellular toxicity. Compound 1 was most effective and can be used as a molecular probe for future studies of how small molecules may affect NDI caused by mutant V2R. These compounds, however, failed to rescue the V2R Y128S mutant, indicating that the compounds described may not work in the rescue of all known mutants of V2R. Taken collectively, the present studies have now identified a promising lead compound that could function as a pharmacoperone to correct the trafficking defect of the NDI-associated mutant V2R L83Q and thus has the therapeutic potential for the treatment of NDI

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias.

Pharmacoperones are small molecules that diffuse into cells and rescue misfolded, mistrafficked protein mutants, restoring their function. These substances act with high target specificity, serving as templates to fold (or refold) receptors, enzymes, ion channels or other proteins and enable them to pass the scrutiny of the cellular quality control system ("rescue"). In the present study we demonstrate that a rescued mutant (L83Q) of the vasopressin type 2 receptor (V2R), shows a strong bias for Gs coupling unlike the WT V2 receptor, which couples to both Gs and Gq/11. Failure of the mutant to couple to Gq/11 was not due to a limiting quantity of G-proteins since other Gq/11-coupled receptors (WT V2R, histamine receptor and muscarinic receptor) responded appropriately to their ligands. Transfection with DNA encoding Gq enabled the V2 receptor mutant to couple to this G protein, but only modestly compared with the WT receptor. Fourteen V2R mutant pharmacoperones, of multiple chemical classes, obtained from a high throughput screen of a 660,000 structure library, and one V2R peptidomimetic antagonist rescues L83Q. The rescued mutant shows similar bias with all pharmacoperones identified, suggesting that the bias is intrinsic to the mutant protein's structure, rather than due to the chemical class of the pharmacoperone. In the case of V2R mutant Y128S, rescue with a pharmacoperone revealed constitutive activity, also with bias for Gs, although both IP and cAMP were produced in response to agonist. These results suggest that particular rescued receptor mutants show functional characteristics that differ from the WT receptor; a finding that may be important to consider as pharmacoperones are developed as therapeutic agents

Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

iv, 60 hlm, : ils, ; 21 c

Observation of J/ψpJ/\psi p Resonances Consistent with Pentaquark States

<p>Transiently transfected HeLa cells containing a total of 100 ng cDNA / 0.125 ml of hWT V2R or the L83Q mutant were treated with different pharmacoperones (10 μM or #50 was 1 μM) to show if the rescued L83Q mutant is coupled to Gs (cAMP). The cells were incubated with the pharmacoperones for 16 h, then washed and stimulated with 1 μM vasopressin containing 0.2 mM IBMX for 30 minutes and the cAMP response was measured. DMSO (vehicle) is a negative control that does not rescue the mutant and SR121463B is a known pharmacoperone for the L83Q mutant. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM).</p

IP production following transient transfection of HeLa cells for selected GPCRs and V2R mutants.

<p>Transiently transfected HeLa cells containing a total of 100 ng cDNA/ 0.125 ml of hWT V2R or the mutant hL83Q were used to compare Gq (IP) coupling with the vasopressin receptor, endogenous muscarinic M3 subtype and histamine H1 receptors with or without 10 μM SR121463B pharmacoperone (rescues the L83Q mutant), DMSO (vehicle) is a negative control. Cells were stimulated with various doses of vasopressin for 2 h, acetylcholine and histamine dihydrochloride for 30 minutes. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p value < 0.05 is considered significant.</p