查看更多>>摘要：The human calcium-sensing receptor (CaSR) is a G protein-coupled receptor that maintains extracellular Ca21 homeostasis by regulating the secretion of parathyroid hormone. Upacicalcet is a novel positive allosteric modulator of CaSR that is used for the treatment of secondary hyperparathyroidism. In the present study, to clarify the binding site of upacicalcet to CaSR, we conducted binding studies and agonistic activity studies in HEK-293T cells expressing human CaSR (intact and mutant) and an in silico docking-simulation analysis. As a result, upacicalcet competed with L-tryptophan and was thought to affect the amino acid binding site. In addition, the effects of substitutions at the amino acid binding site on the binding abilities to upacicalcet as well as the effects on receptor function as measured using inositol-1 mono-phosphate accumulation were examined. Upacicalcet interacted with several CaSR residues that constitute the amino acid binding site. Based on these results, we performed an in silico analysis and obtained a binding mode, consistent with the in vitro study results. Our study revealed that upacicalcet is a novel secondary hyperparathyroidism drug that targets the amino acid binding site of CaSR. Upacicalcet is expected to become a new treatment option for secondary hyperparathyroidism because the binding site differs from that of conventional drugs; consequently, it may be effective for patients who are not sensitive to conventional drugs, and it may have a superior safety profile.
Anuja R. BonyJeffrey R. McArthurAkari KomoriAnn R. Wong...
13页
查看更多>>摘要：The analgesic a-conotoxins Vc1.1, RgIA, and PeIA attenuate noci-ceptive transmission via activation of G protein-coupled GABAb receptors (GABAbRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABABR and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic a-conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABABR subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the a-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABAbR ligand-binding site. The effects of bac-lofen and a-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABAbR mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABAbR subunits attenuated baclofen-sensitive IBa inhibition by the analgesic a-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABAbR residues involved in interactions with the a-conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A 1 S153A, abolished inhibition by both baclofen and the a-conotoxins. Depolarization-activated IBa mediated by both wild-type and all GABAbR mutants were inhibited by the selective GABAbR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic a-conotoxins to the VFT domains of the GABABR.
查看更多>>摘要：Structurally rearranged extrachromosomal circular DNAs (eccDNAs) have been identified in tumor cells, many of which carry regions related to recurrent cancer driver oncogenes (e.g., CCND1, EGFR, and MYC). In a tumor cell, eccDNAs are carrying regions associated with oncogene amplification (>10-fold amplified-copy numbers in human tumors) and poor outcome across multiple cancers. Even though dual-delivery of pairs of CRISPR and CRISPR-associated protein 9 (Cas9) guiding RNAs into normal human cells was reported to induce circulari-zation of genes and chromosomes, in bacteria, the CRISPR-Cas9 system primarily targets extrachromosomal rearranged elements. Likewise, in cancer cells, it is expected that a designed CRISPR-Cas9 system would be able to target extrachromosomal copy number amplifications and produce double strand breaks detrimental to cellular fitness by dictating gene-independent copy number loss-of-fitness effects and antiproliferative responses. A system designed against amplified amplicons may provide a novel approach for cancer therapy and propose a practical implication for CRISPR-Cas9 pairs as a pathway in therapeutic strategies of cancer.
NSTL
Amer Soc Pharmacology Experimental Therapeutics