Ligand-induced
protein degradation has emerged as a compelling
approach to promote the targeted elimination of proteins from cells
by directing these proteins to the ubiquitin-proteasome machinery.
So far, only a limited number of E3 ligases have been found to support
ligand-induced protein degradation, reflecting a dearth of E3-binding
compounds for proteolysis-targeting chimera (PROTAC) design. Here,
we describe a functional screening strategy performed with a focused
library of candidate electrophilic PROTACs to discover bifunctional
compounds that degrade proteins in human cells by covalently engaging
E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs
act through modifying specific cysteines in DCAF11, a poorly characterized
E3 ligase substrate adaptor. We further show that DCAF11-directed
electrophilic PROTACs can degrade multiple endogenous proteins, including
FBKP12 and the androgen receptor, in human prostate cancer cells.
Our findings designate DCAF11 as an E3 ligase capable of supporting
ligand-induced protein degradation via electrophilic PROTACs.
Dermagraft is three-dimensional, allogeneic, human neonatal dermal fibroblast culture grown on a degradable scaffold and cryopreserved. Clinical trials for treatment of diabetic foot ulcers showed optimal healing within a therapeutic range of metabolic activity, determined by 3[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) reduction. Actions of Dermagraft in wound repair include colonization by cells and provision of growth factors and cytokines, both activities dependent on living cells. Cells in the cryopreserved culture showed 60% viability by dye exclusion and, when isolated, were able to proliferate in monolayer culture. Protein synthesis by Dermagraft was inhibited 70-98% by cryopreservation, but, if within the therapeutic range, recovered to 45-85% of the prefreeze value over 48 h. Subtherapeutic Dermagraft showed variable, low recovery. Expression of mRNA for vascular endothelial growth factor (VEGF), platelet-derived growth factor A chain, and insulin-like growth factor-1 was reduced >83% in subtherapeutic compared with therapeutic Dermagraft. Granulocyte colony-stimulating factor and VEGF protein secretion, determined by enzyme-linked immunosorbent assay (ELISA), and angiogenic activity also depended on therapeutic range. VEGF secretion dropped sharply with MTT reductase in subtherapeutic tissue. The data demonstrate the critical dependence of the therapeutic properties of this living dermal implant on recovery of protein synthesis, growth factor expression, and angiogenesis, determined by metabolic activity.
There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42--potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.
Three isoforms of nitric oxide synthase (NOS), dimeric enzymes that catalyze the formation of nitric oxide (NO) from arginine, have been identified. Inappropriate or excessive NO produced by iNOS and/or nNOS is associated with inflammatory and neuropathic pain. Previously, we described the identification of a series of amide-quinolinone iNOS dimerization inhibitors that although potent, suffered from high clearance and limited exposure in vivo. By conformationally restricting the amide of this progenitor series, we describe the identification of a novel series of benzimidazole-quinolinone dual iNOS/nNOS inhibitors with low clearance and sustained exposure in vivo. Compounds were triaged utilizing an LPS challenge assay coupled with mouse and rhesus pharmacokinetics and led to the identification of 4,7-imidazopyrazine 42 as the lead compound. 42 (KD7332) (J. Med. Chem. 2009, 52, 3047 - 3062) was confirmed as an iNOS dimerization inhibitor and was efficacious in the mouse formalin model of nociception and Chung model of neuropathic pain, without showing tolerance after repeat dosing. Further 42 did not affect motor coordination up to doses of 1000 mg/kg, demonstrating a wide therapeutic margin.
Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.
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