nTZDpa

Discovery and Optimization of nTZDpa as an Antibiotic Effective Against Bacterial Persisters.[Pubmed:30132650]

ACS Infect Dis. 2018 Nov 9;4(11):1540-1545.

Conventional antibiotics are not effective in treating infections caused by drug-resistant or persistent nongrowing bacteria, creating a dire need for the development of new antibiotics. We report that the small molecule nTZDpa, previously characterized as a nonthiazolidinedione peroxisome proliferator-activated receptor gamma partial agonist, kills both growing and persistent Staphylococcus aureus cells by lipid bilayer disruption. S. aureus exhibited no detectable development of resistance to nTZDpa, and the compound acted synergistically with aminoglycosides. We improved both the potency and selectivity of nTZDpa against MRSA membranes compared to mammalian membranes by leveraging synthetic chemistry guided by molecular dynamics simulations. These studies provide key insights into the design of selective and potent membrane-active antibiotics effective against bacterial persisters.

Hydrogen/deuterium-exchange (H/D-Ex) of PPARgamma LBD in the presence of various modulators.[Pubmed:16823031]

Protein Sci. 2006 Aug;15(8):1883-92.

A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPARgamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPARgamma ligand binding domain (LBD) with or without molecules that modulate PPARgamma activity. The H/D-Ex patterns of ligand-free PPARgamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPARgamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPARgamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

[Role of angiotensin II receptor antagonists in the treatment of metabolic syndrome].[Pubmed:16762293]

Rev Clin Esp. 2006 Jun;206(6):284-8.

The metabolic syndrome, defined as the association of abdominal obesity, insulin resistance, dyslipidemia and hypertension, is a very prevalent disorder. Moreover, it identifies patients with a high cardiovascular risk, and when diagnosed, life style modifications and/or drug therapy can be initiated in these patients with the aim to reduce their cardiovascular risk. In the last few years, there has been much interest on drugs that lower insulin resistance, a central component of the metabolic syndrome as well as drugs that interrupt the renin-angiotensin system (achieved by angiotensive converting enzyme inhibitors and angiotensin II receptor blockers), due to their beneficial metabolic effects. Of special interest are the so-called selective PPARg modulators, such as telmisartan or the nTZDpa compound. In the future, they may show important benefits in the treatment of patients with the metabolic syndrome.

Regulation of the growth arrest and DNA damage-inducible gene 45 (GADD45) by peroxisome proliferator-activated receptor gamma in vascular smooth muscle cells.[Pubmed:12881480]

Circ Res. 2003 Aug 22;93(4):e38-47.

Peroxisome proliferator-activated receptor (PPAR) gamma is activated by thiazolidinediones (TZDs), widely used as insulin-sensitizing agents for the treatment of type 2 diabetes. TZDs have been shown to induce apoptosis in a variety of mammalian cells. In vascular smooth muscle cells (VSMCs), proliferation and apoptosis may be competing processes during the formation of restenotic and atherosclerotic lesions. The precise molecular mechanisms by which TZDs induce apoptosis in VSMCs, however, remain unclear. In the present study, we demonstrate that the TZDs rosiglitazone (RSG), troglitazone (TRO), and a novel non-TZD partial PPARgamma agonist (nTZDpa) induce caspase-mediated apoptosis of human coronary VSMCs. Induction of VSMC apoptosis correlated closely with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) mRNA expression and transcription, a well-recognized modulator of cell cycle arrest and apoptosis. Using adenoviral-mediated overexpression of a constitutively active PPARgamma mutant and the irreversible PPARgamma antagonist GW9662, we provide evidence that PPARgamma ligands induce caspase-mediated apoptosis and GADD45 expression through a receptor-dependent pathway. Deletion analysis of the GADD45 promoter revealed that a 153-bp region between -234 and -81 bp proximal to the transcription start site, containing an Oct-1 element, was crucial for the PPARgamma ligand-mediated induction of the GADD45 promoter. PPARgamma activation induced Oct-1 protein expression and DNA binding and stimulated activity of a reporter plasmid driven by multiple Oct-1 elements. These findings suggest that activation of PPARgamma can lead to apoptosis and growth arrest in VSMCs, at least in part, by inducing Oct-1-mediated transcription of GADD45. The full text of this article is available online at http://www.circresaha.org.

A non-thiazolidinedione partial peroxisome proliferator-activated receptor gamma ligand inhibits vascular smooth muscle cell growth.[Pubmed:12694805]

Eur J Pharmacol. 2003 Apr 18;466(3):225-34.

Several peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione class inhibit vascular smooth muscle cell proliferation. It is not known whether the antiproliferative activity of PPARgamma agonists is limited to the thiazolidinedione class and/or is directly mediated through PPARgamma-dependent transactivation of target genes. We report here that a novel non-thiazolidinedione partial PPARgamma agonist (nTZDpa) attenuates rat aortic vascular smooth muscle cell proliferation. In a transfection assay for PPARgamma transcriptional activation, the non-thiazolidinedione partial PPARgamma agonist elicited approximately 25% of the maximal efficacy of the full PPARgamma agonist rosiglitazone. In the presence of the non-thiazolidinedione partial PPARgamma agonist, the transcriptional activity of the full agonist, rosiglitazone, was blunted, indicating that the non-thiazolidinedione partial PPARgamma agonist inhibits rosiglitazone-induced PPARgamma activity. The non-thiazolidinedione partial PPARgamma agonist (0.1-10 microM) inhibited vascular smooth muscle cell growth which was accompanied by an inhibition of retinoblastoma protein phosphorylation. Mitogen-induced downregulation of the cyclin-dependent kinase (CDK) inhibitor p27(kip1), and induction of the G1 cyclins cyclin D1, cyclin A, and cyclin E were also attenuated by the non-thiazolidinedione partial PPARgamma agonist. Maximal antiproliferative activity of the non-thiazolidinedione partial PPARgamma agonist required functional PPARgamma as adenovirus-mediated overexpression of a dominant-negative PPARgamma mutant partially reversed its inhibition of vascular smooth muscle cell growth. In contrast, overexpression of dominant-negative PPARgamma did not reverse the inhibitory effect of the non-thiazolidinedione partial PPARgamma agonist on cyclin D1. As the full PPARgamma agonist rosiglitazone exhibited no effect on cyclin D1, inhibition of that G1 cyclin by the non-thiazolidinedione partial PPARgamma agonist likely occurred through a PPARgamma-independent mechanism. These data demonstrate that a non-thiazolidinedione partial PPARgamma agonist may constitute a novel therapeutic for proliferative vascular diseases and could provide additional evidence for the important role of PPARgamma in regulating vascular smooth muscle cell proliferation.

Peroxisome proliferator-activated receptor gamma inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells.[Pubmed:12677008]

Mol Endocrinol. 2003 Jun;17(6):1005-18.

Using a cDNA array consisting only of cell cycle genes, we found that a novel nonthiazolidinedione partial peroxisome proliferator-activated receptor gamma (PPARgamma) agonist (nTZDpa) inhibited expression of minichromosome maintenance (MCM) proteins 6 and 7 in vascular smooth muscle cells. MCM proteins are required for the initiation and elongation stages of DNA replication and are regulated by the transcription factor E2F. Mitogen-induced MCM6 and MCM7 mRNA expression was potently inhibited by nTZDpa and to a lesser degree by the full PPARgamma agonist, rosiglitazone. Inhibition of MCM6 and MCM7 expression by nTZDpa and rosiglitazone paralleled their effect to inhibit phosphorylation of the retinoblastoma protein and cell proliferation. Transient transfection experiments revealed that the nTZDpa inhibited mitogen-induced MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. Adenoviral-mediated E2F overexpression reversed the suppressive effect of nTZDpa on MCM6 and MCM7 expression. Furthermore, activity of a luciferase reporter plasmid driven by multiple E2F elements was inhibited by nTZDpa, indicating that their down-regulation by nTZDpa involves an E2F-dependent mechanism. Overexpression of dominant-negative PPARgamma or addition of a PPARgamma antagonist, GW 9662, blocked nTZDpa inhibition of MCM7 transcription. Adenovirus-mediated overexpression of constitutively active PPARgamma inhibited MCM7 expression in a similar manner as the nTZDpa. These findings provide strong evidence that activation of PPARgamma attenuates MCM7 transcription and support the important role of this nuclear receptor in regulating vascular smooth muscle cell proliferation.

Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator.[Pubmed:12554792]

Mol Endocrinol. 2003 Apr;17(4):662-76.

Antidiabetic thiazolidinediones (TZDs) and non-TZD compounds have been shown to serve as agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we report the identification and characterization of a novel non-TZD selective PPARgamma modulator (nTZDpa). nTZDpa bound potently to PPARgamma with high selectivity vs. PPARalpha or PPARdelta. In cell-based assays for transcriptional activation, nTZDpa served as a selective, potent PPARgamma partial agonist and was able to antagonize the activity of PPARgamma full agonists. nTZDpa also displayed partial agonist effects when its ability to promote adipogenesis in 3T3-L1 cells was evaluated. Assessment of protein conformation using protease protection or solution nuclear magnetic resonance spectroscopy methods showed that nTZDpa produced altered PPARgamma conformational stability vs. full agonists, thereby establishing a physical basis for its observed partial agonism. DNA microarray analysis of RNA from 3T3-L1 adipocytes treated with nTZDpa or several structurally diverse PPARgamma full agonists demonstrated qualitative differences in the affected gene expression profile for nTZDpa. Chronic treatment of fat-fed, C57BL/6J mice with nTZDpa or a TZD full agonist ameliorated hyperglycemia and hyperinsulinemia. However, unlike the TZD, nTZDpa caused reductions in weight gain and adipose depot size. Feed efficiency was also substantially diminished. Unlike TZDs, nTZDpa did not cause cardiac hypertrophy in mice. When a panel of PPARgamma target genes was examined in white adipose tissue, nTZDpa produced a different in vivo expression pattern vs. the full agonist. These findings establish that novel selective PPARgamma modulators can produce altered receptor conformational stability leading to distinctive gene expression profiles, reduced adipogenic cellular effects, and potentially improved in vivo biological responses. Such compounds may lead to preferred therapies for diabetes, obesity, or metabolic syndrome.