Kinetic characterization and identification of a novel inhibitor of hypoxia-inducible factor prolyl hydroxylase 2 using a time-resolved fluorescence resonance energy transfer-based assay technology.

The human hypoxia-inducible factor prolyl hydroxylases 1, 2, and 3 (HIF-PHD1, -2, and -3) are thought to act as proximal sensors of cellular hypoxia by virtue of their mechanism-based dependence on molecular oxygen. These 2-oxoglutarate (2-OG) and non-heme iron-dependent oxygenases constitutively hydroxylate HIF, resulting in high-affinity binding to Von Hippel-Lindau protein (pVHL). Some reported affinities for the HIF-PHDs for 2-OG and iron approach the estimated physiological concentrations for these cofactors, suggesting that the system as described is not catalytically optimal. Here we report the enzymatic characterization of full-length recombinant human HIF-PHD2 using a novel and sensitive catalytic assay. We demonstrated submicromolar affinities for 2-OG and ferrous iron and HIF-PHD2 Km values for oxygen that are greater than atmospheric oxygen levels, suggesting that molecular oxygen is indeed the key regulator of this pathway. In addition, we observed enhancement of HIF-PHD2 catalytic activity in the presence of ascorbic acid with only minor modifications of HIF-PHD2 requirements for 2-OG, and a detailed pH study demonstrated optimal HIF-PHD2 catalytic activity at pH 6.0. Lastly, we used this sensitive and facile assay to rapidly perform a large high-throughput screen of a chemical library to successfully identify and characterize novel 2-OG competitive inhibitors of HIF-PHD2.

Structure-guided design of substituted aza-benzimidazoles as potent hypoxia inducible factor-1alpha prolyl hydroxylase-2 inhibitors.

We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.

Discovery of novel hydroxy-thiazoles as HIF-alpha prolyl hydroxylase inhibitors: SAR, synthesis, and modeling evaluation.

Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.

Structure-guided Discovery of Dual-recognition Chemibodies.

Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.

360-Degree Trabeculotomy for Medically Refractory Glaucoma Following Cataract Surgery and Juvenile Open-Angle Glaucoma.

PURPOSE: Although angle surgeries show good success in primary congenital glaucoma, reported success in glaucoma following cataract surgery (GFCS) and juvenile open-angle glaucoma (JOAG) is variable and with relatively short follow-up. We evaluated longer-term outcomes of 360-degree trabeculotomy for medically refractory GFCS and JOAG. DESIGN: Retrospective case series. METHODS: First operated eyes of consecutive patients with medically refractory GFCS and JOAG in a single-surgeon pediatric glaucoma practice who underwent illuminated microcatheter-assisted 360-degree trabeculotomy from February 2008 to June 2015 were reviewed. Baseline characteristics, time to failure or last visit, surgical details, final intraocular pressure (IOP), and complications were recorded. Success required IOP ≤22 mm Hg and 20% reduction without additional glaucoma surgery or devastating complication. RESULTS: Thirty-five eyes (35 patients) were included: 25 GFCS and 10 JOAG (mean age at surgery 5.6 vs 16.7 years, respectively, P < .001). Success for GFCS and JOAG was 18 of 25 (72%) vs 6 of 10 (60%) eyes at mean follow-up of 31.9 ± 26.1 vs 24.5 ± 19.7 months, respectively. IOP was significantly reduced from baseline for both GFCS and JOAG (31.5 ± 7.5 mm Hg vs 19.2 ± 7.7 mm Hg, P < .001; and 29.5 ± 10.3 mm Hg vs 15.8 ± 6.6 mm Hg, P < .001, respectively). Fewer glaucoma medications were needed after surgery (P = .01) for GFCS but not JOAG. Complications (all but 2 spontaneously resolving) included choroidal effusion (1), vitreous hemorrhage (3), Descemet detachment (1), and persistent hyphema (2). Three-year Kaplan-Meier success for GFCS vs JOAG was 75.3% vs 53.3%, respectively. CONCLUSIONS: Illuminated microcatheter-assisted 360-degree trabeculotomy is a useful, low-risk, modestly successful initial surgical treatment for both medically refractory GFCS and JOAG.

Discovery of 1H-pyrazol-3(2H)-ones as potent and selective inhibitors of protein kinase R-like endoplasmic reticulum kinase (PERK).

The structure-based design and optimization of a novel series of selective PERK inhibitors are described resulting in the identification of 44 as a potent, highly selective, and orally active tool compound suitable for PERK pathway biology exploration both in vitro and in vivo.

Illuminated microcatheter-facilitated 360-degree trabeculotomy for refractory aphakic and juvenile open-angle glaucoma.

BACKGROUND: Aphakic and juvenile open-angle glaucoma (JOAG) cases often prove to be challenging to manage, frequently requiring surgical intervention. Angle surgery has some reported success in these cases. PURPOSE: The purpose of this study was to evaluate 360-degree trabeculotomy, facilitated by iTrack, for refractory aphakic glaucoma and JOAG. PATIENTS AND METHODS: This study was conducted to evaluate the success and complication rates of illuminated microcatheter-assisted 360-degree trabeculotomy for aphakic glaucoma and JOAG (2 surgeons/2 sites, 2008 to 2011). The success of this surgery was defined as intraocular pressure ≤22 mm Hg with >30% reduction, without disease progression, oral glaucoma medications, or additional glaucoma surgery. One eye per subject was analyzed. All had gonioscopically open angles preoperatively. RESULTS: A total of 23 eyes status post iTrack-facilitated 360-degree trabeculotomy, 13 aphakic glaucoma cases (mean age 3.1 y at surgery), and 10 JOAG cases (mean age, 18.6 y) were included in the study. Complete cannulation/opening of the Schlemm canal occurred intraoperatively in 8 aphakic and in all JOAG cases. Success rates achieved at last follow-up were as follows: 8/13 (62%) aphakic glaucoma cases and 9/10 (90%) JOAG cases. Preoperative versus final intraocular pressure decreased for all surgically successful eyes (35.5±3.9 vs. 17.3±4.6 mm Hg for aphakic glaucoma, P<0.0001, after mean 30 mo and 30.7±7.4 vs. 13.4±2.8 mm Hg for JOAG, P=0.0001, after mean 10 mo). All trabeculotomy failures (n=5) occurred within 5 months. Complications included vitreous hemorrhage (2 aphakic eyes) and transient choroidal effusion (1 aphakic eye). CONCLUSIONS: iTrack-facilitated 360-degree trabeculotomy shows early promise for initial surgical treatment of medically refractory aphakic glaucoma and JOAG, with few complications and without affecting future surgical options.

Synthesis and structure-activity relationship of trisubstituted thiazoles as Cdc7 kinase inhibitors.

The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors.

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.