Joseph Clemas

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

A PPARγ mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

Identification of Leu276 of the S1P1 Receptor and Phe263 of the S1P3 Receptor in Interaction with Receptor Specific Agonists by Molecular Modeling, Site-Directed Mutagenesis, and Affinity Studies

Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P1 against S1P3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P1 and S1P3 receptors. Computational models of human S1P1 and S1P3 receptors bound with two nonselective agonists or two S1P1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P1 Leu276 and S1P3 Phe263 contribute to the S1P1/S1P3 selectivity of the two S1P1-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P1-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P1 and S1P3 receptors, which should aid development of more selective S1P1 receptor agonists with immunosuppressive properties and improved safety profiles.

Mineralocorticoid receptor antagonists: Identification of heterocyclic amide replacements in the oxazolidinedione series

Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure-activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists.

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.

Discovery of benzimidazole oxazolidinediones as novel and selective nonsteroidal mineralocorticoid receptor antagonists.

Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.

Discovery of Spirocyclic Aldosterone Synthase Inhibitors as Potential Treatments for Resistant Hypertension.

Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.

Discovery of novel non-steroidal reverse indole mineralocorticoid receptor antagonists.

Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.