Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107.

Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca(2+) from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12) increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine derivative JTV519, and the more specific derivative S107 (2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report a high degree of nonspecific and specific low affinity [(3)H]S107 binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [(3)H]S107 per RyR1 tetramer with EC(50) ∼52 µM and Hillslope ∼2. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of reduced glutathione and the NO-donor NOC12, with no effect in the presence of oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12 dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 µM myoplasmic FKBP12 and was attenuated by S107. In single channel measurements with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca(2+) release in skeletal muscle by binding to RyR1 low affinity sites.

Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.

Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates.

ADP-ribosyl cyclase and NAD+ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide mononucleotide (NMN+) with an alternative base. 4'-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k(cat)/K(M)=0.9-10 microM(-1)s(-1)) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k(cat)/K(M) values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 microM(-1)s(-1)). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN+, k(cat)/K(M)=0.05-0.4 microM(-1)s(-1). The k(cat)/K(M) values for the exchange reaction with NMN+ were generally similar (0.04-0.12 microM(-1)s(-1)) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k(cat)/K(M) values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.