Institute for Diabetes Discovery

Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 μm and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 μm) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo.

Cerebral oximetry using near-infrared spectroscopy is a noninvasive optical technology to detect cerebral hypoxia-ischemia and develop interventions to prevent and ameliorate hypoxic brain injury. Cerebral oximeters are calibrated and validated by comparison of the near-infrared spectroscopy-measured cerebral O2 saturation (SctO2) to a "field" or reference O2 saturation (REF CX) calculated as a weighted average from arterial and jugular bulb oxygen saturations. In this study, we calibrated and validated the second-generation, 5 wavelength, FORE-SIGHT Elite with the medium sensor (source-detector separation 12 and 40 mm) for measurement of SctO2 in children with congenital heart disease. After institutional review board approval and written informed consent, 63 children older than 1 month and ≥2.5 kg scheduled for cardiac catheterization were enrolled. Self-adhesive FORE-SIGHT Elite medium sensors were placed on the right and left sides of the forehead. Blood samples for calculation of REF CX were drawn simultaneously from the aorta or femoral artery and the jugular bulb before (T1) and shortly after (T2) baseline hemodynamic measurements. FORE-SIGHT Elite SctO2 measurements were compared to the REF CX (REF CX = [0.3 SaO2] + [0.7 SjbO2]) using Deming regression, least squares linear regression, and Bland-Altman analysis. Sixty-one subjects (4.5 [standard deviation 4.4] years of age; 17 [standard deviation 13] kg, male 56%) completed the study protocol. Arterial oxygen saturation ranged from 64.7% to 99.1% (median 96.0%), jugular bulb venous oxygen saturation from 34.1% to 88.1% (median 68.2%), the REF CX from 43.8% to 91.4% (median 76.9%), and the SctO2 from 47.8% to 90.8% (median 76.3%). There was a high degree of correlation in SctO2 between the right and left sensors at a given time point (within subject between sensor correlation r = 0.91 and 95% confidence interval [CI], 0.85-0.94) or between T1 and T2 for the right and left sensors (replicates, within subject between time point correlation r = 0.95 and 95% CI, 0.92-0.96). By Deming regression, the estimated slope was 0.966 (95% CI, 0.786-1.147; P = .706 for testing against null hypothesis of slope = 1) with a y intercept of 2.776 (95% CI, -11.102 to 16.654; P = .689). The concordance correlation coefficient was 0.873 (95% CI, 0.798-0.922). Bland-Altman analysis for agreement between SctO2 and REF CX that accounted for repeated measures (both in times and sensors) found a bias of -0.30% (95% limits of agreement: -10.56% to 9.95%). This study calibrated and validated the FORE-SIGHT Elite tissue oximeter to accurately measure SctO2 in pediatric patients with the medium sensor.

In this work, the quantitative analysis of phytochemical constituents of medicinal plant Spiraea hypericifolia L. from Kazakhstan has been made for the first time. Total bioactive components aerial and underground parts of plants material S. hypericifolia L. such as organic acids (0.28 %), (0.35 %), flavonoids (0.77 %), (5.36 %), coumarins (0.04 %), (0.21 %), saponins (2.09 %), (0.98 %), tannins (1.31 %), (1.62 %), and together with moisture content (4.72 %), (3.84 %), total ash (5.11 %), (5.41 %), were determined. Eleven macro-micro elements from the ash of plant were identified, main contents of them were Ca (362.20 g/ml), (344.750 g/ml), K (69.670 g/ml), (57.8075 g/ml), and Mg (24.270 g/ml), (34.480 g/ml), by using the method of multi-element atomic emission spectral analysis. In addition, twenty amino and eight fatty acids were analyzed from the plant. The results showed that major contents of amino acids were glutamate (1741 mg/100g), (1710 mg/100g), aspartate (845 mg/100g), (820 mg/100g), and alanine (560 mg/100g), (523 mg/100g), as well as in fatty acid was linoleic (81.1 %), (79.7 %) acid, respectively.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

The soluble mycobacterial carbonic anhydrases Rv3588c and Rv1284 belong to a different class of carbonic anhydrases than those found in humans, making them attractive drug targets by using the inherent differences in the folds of the different classes. By screening a natural product library, we identified naphthoquinone derivatives as a novel non-classical inhibitor scaffold of mycobacterial carbonic anhydrases that lack the sulfonamide/sulfamate group and thus did not affect human carbonic anhydrase II.

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

Assessment of a new chemical entity for cytochrome P450 (CYP) enzyme induction at an early stage in discovery is crucial to prevent potential drug-drug interactions. CYP3A, the most abundant CYP isoform in the liver, metabolizes approximately 50% of drugs currently on the market and is also a highly inducible enzyme. The use of both rat and human hepatocyte culture for the prediction of in vivo CYP3A induction has become refined and validated and is considered a standard in vitro model. The current evaluation of CYP3A enzyme induction involves the use of substrates requiring subsequent analysis of metabolites by high-performance liquid chromatography/mass spectrometry, which adds considerable time and cost. In the present study, we describe the use of a novel luminogenic substrate, luciferin-6'-pentafluoro-benzyl ether (PFBE), which allows for a fast and selective measurement of CYP3A enzyme induction in cultured rat hepatocytes. The extent of induction was evaluated using cells treated for 3 d with the prototypical inducers, dexamethasone, phenobarbital, and pregnenolone 16 alpha-carbonitrile (PCN). Enzyme activity was measured in the treated cells either by the depentafluorobenzylation of luciferin-PFBE or the testosterone 6-beta-hydroxylation. Using both methods, dexamethasone and PCN-treated cells exhibited strong CYP3A activity, whereas phenobarbital treatment resulted in a weak response. The fold induction varied between both methods, but this variability can be controlled by normalizing data from each treatment to a positive control. The results indicate that luciferin-PFBE is an attractive alternative to the use of conventional substrate, testosterone, providing a sensitive, robust, and rapid method compatible with the multiwell plate format for the assessment of CYP3A induction.

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 angstrom, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2(1)), IDD 393 (resolution: 0.90 angstrom, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 angstrom, IC50: 9 nM, space group: P2(1)) and minalrestat (resolution: 1.10 angstrom, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 approximately 0.3 angstrom). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl(-) ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, IDD384, has been solved at 1.7 A resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitor's hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pK(a) = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitor's polar head depends on its protonation state.