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- Influence of Tyrosine Kinase Inhibition on Organic Anion Transporting
Polypeptide 1B3-Mediated Uptake [Articles]-
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Hove, V. N; Anderson, K, Hayden, E. R, Pasquariello, K. Z, Gibson, A. A, Shen, S, Qu, J, Jin, Y, Miecznikowski, J. C, Hu, S, Sprowl, J. A. Pages: 381 - 389 Abstract: The organic anion transporting polypeptide family member (OATP) 1B3 is a hepatic uptake transporter that has a broad substrate recognition and plays a significant role in regulating elimination of endogenous biomolecules or xenobiotics. OATP1B3 works in tandem with OATP1B1, with which it shares approximately 80% sequence homology and a high degree of substrate overlap. Despite some substrates being recognized solely by OATP1B3, its ability to compensate for loss of OATP1B1-mediated elimination and recognition by regulatory agencies, little is known about OATP1B3 regulatory factors and how they are involved with drug-drug interaction. It was recently discovered that OATP1B1 function is mediated by the activity of a particular tyrosine kinase that is sensitive to a variety of tyrosine kinase inhibitors (TKIs). This study reports that OATP1B3 is similarly regulated, as at least 50% of its activity is reduced by 20 US Food and Drug Administration -approved TKIs. Nilotinib was assessed as the most potent OATP1B3 inhibitor among the investigated TKIs, which can occur at clinically relevant concentrations and acted predominantly through noncompetitive inhibition without impacting membrane expression. Finally, OATP1B3 function was determined to be sensitive to the knockdown of the Lck/Yes novel tyrosine kinase that is sensitive to nilotinib and has been previously implicated in mediating OATP1B1 activity. Collectively, our findings identify tyrosine kinase activity as a major regulator of OATP1B3 function which is sensitive to kinase inhibition. Given that OATP1B1 is similarly regulated, simultaneous disruption of these transporters can have drastic effects on systemic drug concentrations, which would promote adverse events.SIGNIFICANCE STATEMENTThe organic anion transporting polypeptide family member (OATP) 1B3 is a facilitator of hepatic drug elimination, although much is unknown of how OATP1B3 activity is mediated, or how such regulators contribute to drug-drug interactions. This study reports that OATP1B3 activity is dependent on the Lck/Yes novel tyrosine kinase, which is sensitive to numerous tyrosine kinase inhibitors. These findings provide insight into the occurrence of many clinical drug-drug interactions, and a rationale for future study of tyrosine kinases regulating drug disposition. PubDate: 2022-05-06T08:00:10-07:00 DOI: 10.1124/molpharm.121.000287 Issue No: Vol. 101, No. 6 (2022)
- Coassembly of Warm Temperature-Sensitive Transient Receptor Potential
Vanilloid (TRPV) 3 and TRPV4 Channel Complexes with Distinct Functional Properties [Articles]-
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Hu, F; Cao, X, Niu, C, Wang, K. Pages: 390 - 399 Abstract: Heteromeric assembly of temperature-sensitive transient receptor potential (TRP) ion channels has been suggested to underlie the molecular basis of fine-tuning of temperature detection and chemical sensation. However, whether warm temperature–sensitive TRP vanilloid (TRPV) 3 and TRPV4 channels robustly expressed in the skin can form heteromeric assembly remains largely unknown. In this study, we show that TRPV3 and TRPV4 channels can coassemble into functional heterotetrameric channels with distinct properties. Confocal imaging reveals a colocalization and association of TRPV3 and TRPV4 proteins in cell membrane. Coimmunoprecipitation analysis demonstrates a strong protein-protein interaction between TRPV3 and TRPV4 subunits from heterogeneously expressed cells or mouse skin tissues through their C termini but not in TRPV3 knockout tissues. Coexpression of TRPV3 and TRPV4 channels yields a heterotetrameric channel complexes characterized by an intermediate single-channel conductance, distinct activation threshold, and pharmacology. Taken together, our findings demonstrate a heterotetrameric assembly of TRPV3 and TRPV4 channels, which may help explain the role of temperature-sensitive TRPV channels in fine-tuning of environmental detection and sensation in the skin.SIGNIFICANCE STATEMENTThe coassembly of transient receptor potential vanilloid (TRPV) 3 and TRPV4 channel complexes increases the functional diversity within the channel subfamily, which may serve as a molecular basis for fine-tuning of environmental detection and temperature sensation in mammals. PubDate: 2022-05-06T08:00:10-07:00 DOI: 10.1124/molpharm.121.000370 Issue No: Vol. 101, No. 6 (2022)
- Secretin Amino-Terminal Structure-Activity Relationships and Complementary
Mutagenesis at the Site of Docking to the Secretin Receptor [Articles]-
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Milburn, J. E; Harikumar, K. G, Piper, S. J, Raval, S, Christopoulos, A, Wootten, D, Sexton, P. M, Miller, L. J. Pages: 400 - 407 Abstract: Class B1 G protein–coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His1 and Ser2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser2 was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7.SIGNIFICANCE STATEMENTThis work refines our molecular understanding of the activation mechanisms of class B1 G protein–coupled receptors. The amino-terminal region of secretin interacts with the seventh transmembrane segment of its receptor with structural specificity and with a charge-charge interaction helping to drive functional activation. PubDate: 2022-05-06T08:00:10-07:00 DOI: 10.1124/molpharm.122.000502 Issue No: Vol. 101, No. 6 (2022)
- Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum
Phosphate and Improve Skeletal Abnormalities in Hyp Mice [Articles]-
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Xiao, Z; Liu, J, Liu, S.-H, Petridis, L, Cai, C, Cao, L, Wang, G, Chin, A. L, Cleveland, J. W, Ikedionwu, M. O, Carrick, J. D, Smith, J. C, Quarles, L. D. Pages: 408 - 421 Abstract: Excess fibroblast growth factor (FGF) 23 causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the fibroblast growth factor receptor/α-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design, we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogs with enhanced drug-like properties. In this study, we tested in a preclinical Hyp mouse homolog of XLH a direct connect analog [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (8n), which exhibited the greatest stability in microsomal assays, and [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] (13a), which exhibited increased in vitro potency. Using cryo-electron microscopy structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541, and its analogs (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation-fluorescence resonance energy transfer assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders.SIGNIFICANCE STATEMENTThis study used structure-based drug design and medicinal chemistry approaches to identify and optimize small molecules with different stability and potency, which antagonize excessive actions of fibroblast growth factor 23 (FGF23) in hereditary hypophosphatemic rickets. The findings confirmed that these antagonists bind to the N-terminus of FGF23 to inhibit its binding to and activation of the fibroblast growth factor receptors/α-Klotho signaling complex. Administration of these lead compounds improved phosphate homeostasis and abnormal skeletal phenotypes in a preclinical Hyp mouse model. PubDate: 2022-05-12T06:38:45-07:00 DOI: 10.1124/molpharm.121.000471 Issue No: Vol. 101, No. 6 (2022)
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