A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells.

The Kinetic Intra-Cellular Assay (KICA) is a recombinant cell-based technique that utilizes NanoBRET technology. KICA enables the measurement of intracellular binding kinetics. This protocol describes steps for cellular transfection and expression, followed by addition of a target specific fluorophore conjugated probe and a range of concentrations of competitor compounds, followed by the measurement of BRET in a 384 well format. Fitting the BRET data allows measurement of forward and reverse binding rates and the determination of KD. For complete details on the use and execution of this profile, please refer to Lay et al. (2021).

Development of an intracellular quantitative assay to measure compound binding kinetics.

Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium-derived measurements of IC50, EC50, or KD. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype; however, these parameters are yet to be fully adopted in early drug discovery. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. Here, we describe an intracellular NanoBRET competition assay that can be used to quantify cellular kinetic rates of compound binding to nanoluciferase-fused bromodomain and extra-terminal (BET) proteins. Comparative rates are generated using a cell-free NanoBRET assay and by utilizing orthogonal recombinant protein-based methodologies. A screen of known pan-BET inhibitors is used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.

Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions.

Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.

Do TRPV1 antagonists increase the risk for skin tumourigenesis? A collaborative in vitro and in vivo assessment.

A recent hypothesis suggesting that the pharmacological target TRPV1 (transient receptor potential vanilloid subfamily, member 1) may function as a tumour suppressor, which potentially impacts the development of TRPV1 antagonist therapeutics for a range of conditions. However, little is known about the long-term physiologic effects of TRPV1 blockade in the skin. In vitro and in vivo studies suggested that the potent TRPV1 competitive antagonist AMG-9810 promoted proliferation in N/TERT1 cells (telomerase-immortalised primary human keratinocytes 1) and tumour development in mouse skin that was mediated through EGFR/Akt/mTOR signalling. We attempted to reproduce the reported in vitro and in vivo findings to further explore this hypothesis to understand the underlying mechanism and the risk associated with TRPV1 antagonism in the skin. In vitro proliferation studies using multiple methods and topical application with AMG-9810 and structurally similar TRPV1 antagonists such as SB-705498 and PAC-14028 were performed. Although we confirmed expression of TRPV1 in primary human epidermal keratinocytes (HEKn) and spontaneously immortalised human keratinocytes (HaCaT), we were unable to demonstrate cell proliferation in either cell type or any clear evidence of increased expression of proteins in the EGFR/Akt/mTOR signalling pathway with these molecules. We were also unable to demonstrate skin tumour promotion or underlying molecular mechanisms involved in the EGFR/Akt/mTOR signalling pathway in a single-dose and two-stage carcinogenesis mouse study treated with TRPV1 antagonists. In conclusion, our data suggest that inhibiting the pharmacological function of TRPV1 in skin by specific antagonists has not been considered to be indicative of skin tumour development.

Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2).

The H(+)-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[(3)H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC(50) 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.

Vigabatrin transport across the human intestinal epithelial (Caco-2) brush-border membrane is via the H+ -coupled amino-acid transporter hPAT1.

The aim of this investigation was to determine if the human proton-coupled amino-acid transporter 1 (hPAT1 or SLC36A1) is responsible for the intestinal uptake of the orally-administered antiepileptic agent 4-amino-5-hexanoic acid (vigabatrin). The Caco-2 cell line was used as a model of the human small intestinal epithelium. Competition experiments demonstrate that [3H]GABA uptake across the apical membrane was inhibited by vigabatrin and the GABA analogues trans-4-aminocrotonic acid (TACA) and guvacine, whereas 1-(aminomethyl)cyclohexaneacetic acid (gabapentin) had no affect. Experiments with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded Caco-2 cells demonstrate that apical exposure to vigabatrin and TACA induce comparable levels of intracellular acidification (due to H+/amino-acid symport) to that generated by GABA, suggesting that they are substrates for a H+ -coupled absorptive transporter such as hPAT1. In hPAT1 and mPAT1-expressing Xenopus laevis oocytes [3H]GABA uptake was inhibited by vigabatrin, TACA and guvacine, whereas gabapentin failed to inhibit [3H]GABA uptake. In Na+ -free conditions, vigabatrin and TACA evoked similar current responses (due to H+/amino-acid symport) in hPAT1-expressing oocytes under voltage-clamp conditions to that induced by GABA (whereas no current was observed in water-injected oocytes) consistent with the ability of these GABA analogues to inhibit [3H]GABA uptake. This study demonstrates that hPAT1 is the carrier responsible for the uptake of vigabatrin across the brush-border membrane of the small intestine and emphasises the therapeutic potential of hPAT1 as a delivery route for orally administered, clinically significant GABA-related compounds.

Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2).

Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate-evoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. Amino (and imino) acid transport via rPAT2 was pH-dependent, Na(+)-independent and electrogenic. At extracellular pH 5.5 (in Na(+)-free conditions) proline uptake was saturable (Km 172+/-41 muM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter.PAT2 preferred substrates are L-alpha-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4- or 5-membered heterocyclic amino and imino acids such as 2-azetidine-carboxylate, proline and cycloserine, where both D- and L-enantiomers are transported. The major restrictions on transport are side chain size (the ethyl group of alpha-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH(2) groups, as in beta-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded. The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2.

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries.

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.