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1.  RUC-4: A Novel αIIbβ3 Antagonist for Pre-hospital Therapy of Myocardial Infarction 
Treatment of myocardial infarction (MI) within the first 1–2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbβ3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbβ3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly (IM) by autoinjector to facilitate its use in the pre-hospital setting. Here we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models.
Approach and Results
RUC-4 was ~20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60–80 mg/ml). It shared RUC-2’s specificity for αIIbβ3 vs αVβ3, did not prime the receptor to bind fibrinogen, or induce changes in β3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human, but not mouse platelets. IM injection of RUC-4 in non-human primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5–24 hours.
RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a pre-hospital therapy of MI, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.
PMCID: PMC4180209  PMID: 25147334
αIIbβ3; platelet; myocardial infarction
2.  Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication by Blocking Viral Ribonuclease H Activity 
Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] = 25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.
PMCID: PMC4335860  PMID: 25451058
3.  A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor 
A collection of αIIbβ3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents’ structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.
PMCID: PMC3951875  PMID: 24461295
4.  CD28 and ITK signals regulate autoreactive T cell trafficking 
Nature medicine  2013;19(12):1632-1637.
Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the coinhibitory receptor CTLA-4 develop fatal autoimmunity characterized by massive lymphocytic invasion into non-lymphoid tissues. Here we demonstrate that the CD28 costimulatory pathway regulates the trafficking of self-reactive Ctla4−/− T cells to tissues. Co-ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation, but instead causes self-reactive Ctla4−/− T cells to accumulate in secondary lymphoid organs. Despite a fulminant autoimmune process in the lymphoid compartment, Itk−/−Ctla4−/− mice are otherwise healthy and exhibit a long lifespan. We propose that ITK licenses autoreactive T cells to enter tissues to mount destructive immune responses. Importantly, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of Type I diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.
PMCID: PMC4005518  PMID: 24270545
5.  Multiple A2E treatments lead to melanization of rod outer segment–challenged ARPE-19 cells 
Molecular Vision  2014;20:285-300.
Daily phagocytosis of outer segments (OSs) and retinoid recycling by the RPE lead to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and bisretinoids such as A2E and its oxidation products. Accumulation of A2E and its oxidation products is implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in the RPE during normal aging. In this study, we used a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation.
To distinguish between pathologic and normal responses of the RPE to A2E accumulation, we treated established ARPE-19 cells (cultured for 3 weeks after reaching confluence) with low micromolar amounts of A2E for several weeks. We compared the lysosomal function, lysosomal pH, degree of OS digestion, and melanization of the treated cells to untreated control cells in response to a challenge of purified rod OSs (ROSs). A2E was analyzed with high-performance liquid chromatography (HPLC); and A2E and melanin were identified with mass spectrometry.
We found that post-confluent ARPE-19 cells took up and accumulated A2E under dim light conditions. Spectral analysis of the HPLC separations and mass spectrometry showed that A2E-fed cells contained A2E and oxidized A2E (furan-A2E). A2E accumulation led to a modest increase (up to 0.25 unit) in lysosomal pH in these cells. The specific activity of cathepsin D and lysosomal acid phosphatase was reduced in the A2E-treated cells, but ROS degradation was not impaired. We found that, upon challenge with ROSs, melanin pigment was induced in the lysosomal fraction of the A2E-treated ARPE-19 cells. Thus, the ARPE-19 cells responded to the A2E treatment and ROS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing.
We used a modified ARPE-19 cell model in which melanization was elicited as a response to chronic accumulation of A2E. We found that although A2E treatment led, as has been previously reported, to modest lysosomal alkalinization and lysosomal impairment of ARPE-19 cells, a potential homeostatic mechanism may involve production of a special type of lysosomes containing melanin.
PMCID: PMC3955416  PMID: 24644403
6.  Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis 
Nature chemical biology  2012;8(10):839-847.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.
PMCID: PMC3711671  PMID: 22922757
7.  ‘Reversine’ and its 2-Substituted Adenine Derivatives as Potent and Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  2005;48(15):4910-4918.
The dedifferentiation agent ‘reversine’ (2-(4-morpholinoanilino)-N6-cyclohexyladenine 2) was found to be a moderately potent antagonist for the human A3 adenosine receptor (AR) with a Ki value 0.66 μM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-phenylamino-N6-(cyclohexyl)adenine 12, 2-phenylamino-N6-(cycloheptyl)adenine 19, and 2-phenylamino-N6-(endo-norbornyl)adenine 21 as potent A3 AR ligands with Ki values of 51, 42 and 37 nM, respectively, with 30 – 200-fold selectivity in comparison to A1 and A2A ARs. The most selective A3 AR antagonist (>200-fold) was 2-phenyloxy-N6-(cyclohexyl)adenine 22. 9-Methylation of 12, but not 19, was well tolerated in A3 AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-phenylamino, 2-phenyloxy, and 2-phenylthio substitutions, the order of affinity at the A3 AR was oxy ≥ amino > thio. Selected derivatives, including reversine (KB value of 466 nM in Schild analysis), competitively antagonized the functional effects of a selective A3 AR agonist, i.e. inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N6 position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A3 AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A3 AR antagonist effects and dedifferentiation.
PMCID: PMC3474371  PMID: 16033270
8.  Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses 
Science (New York, N.Y.)  2011;334(6060):1278-1283.
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
PMCID: PMC3471535  PMID: 22052977
9.  Synthesis, Activity and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H 
Journal of medicinal chemistry  2011;54(13):4462-4473.
The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.
PMCID: PMC3133734  PMID: 21568335
10.  Chiral Kinase Inhibitors 
Small molecule kinase inhibitors are important tools for studying cellular signaling pathways, phenotypes and are, occasionally, useful clinical agents. With stereochemistry pervasive throughout the molecules of life it is no surprise that a single stereocenter can bestow a ligand with distinct binding affinities to various protein targets. While the majority of small molecule kinase inhibitors reported to date are achiral, a number of asymmetric compounds show great utility as tools for probing kinase-associated biomolecular events as well as promising therapeutic leads. The mechanism by which chirality is introduced varies but includes screening of chiral libraries, incorporation of chiral centers during optimization efforts and the rational installation of a chiral moiety as guided by structural and modeling efforts. Here we discuss several advanced chiral small molecule kinase inhibitors where stereochemistry plays an important role in terms of potency and selectivity.
PMCID: PMC3220195  PMID: 21291394
11.  Evaluation of Thieno[3,2-b]pyrrole[3,2-d]pyridazinones as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.
PMCID: PMC2874658  PMID: 20451379
Warburg effect; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
12.  Evaluation of Substituted N,N′-Diarylsulfonamides as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2
PMCID: PMC2818804  PMID: 20017496
Warburg effect; pyruvate kinase; cellular metabolism; high-throughput screening; small molecule activators
14.  Proteasome Inactivation Promotes p38 Mitogen-activated Protein Kinase-dependent Phosphatidylinositol 3-kinase Activation and Increases Interleukin-8 Production in Retinal Pigment Epithelial Cells 
Molecular Biology of the Cell  2009;20(16):3690-3699.
Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. We have demonstrated previously that oxidative inactivation of the proteasome is a molecular link between oxidative stress and overexpression of interleukin (IL)-8. Here, we elucidated a novel signaling cascade that leads to up-regulation of IL-8 in response to proteasome inactivation. The sequence of events in this cascade includes proteasome inactivation, activation of mitogen-activated protein kinase kinase (MKK)3/MKK6, activation of p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor phosphorylation, phosphatidylinositol 3-kinase (PI3K) activation and increased IL-8 expression. Blocking any of these signaling pathways abolished the up-regulation of IL-8 induced by proteasome inhibition. Although Akt is also activated in response to proteasome inactivation, we found that the PI3K-dependent up-regulation of IL-8 is independent of 3-phosphoinositide-dependent protein kinase (PDK)1 and Akt. Inhibition of PDK1 and Akt with chemical inhibitors or expression of constitutive active Akt had little effects on IL-8 expression in response to proteasome inactivation. In contrast, inhibition of interleukin 2-inducible T cell kinase, a kinase downstream of PI3K, significantly reduced the expression and secretion of IL-8 in response to proteasome inactivation. Together, these data elucidate a novel signaling network that leads to increased IL-8 production in response to proteasome inactivation.
PMCID: PMC2777929  PMID: 19570915
15.  Examining the Chirality, Conformation and Selective Kinase Inhibition of 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile (CP-690,550) 
Journal of medicinal chemistry  2008;51(24):8012-8018.
Here, we examine the significance that stereochemistry plays within the clinically relevant Janus Kinase 3 (Jak3) inhibitor CP-690,550. A synthesis of all four enantiopure stereoisomers of the drug was carried out and an examination of each compound revealed that only the enantiopure 3R, 4R isomer was capable of blocking Stat5 phosphorylation (Jak3 dependent). Each compound was profiled across a panel of over 350 kinases which revealed a high level of selectivity for the Jak family kinases for these related compounds. Each stereoisomer retained a degree of binding to Jak3 and Jak2 and the 3R, 4S and 3S, 4R stereoisomers were further revealed to have binding affinity for selected members of the STE7 and STE20 subfamily of kinases. Finally, an appraisal of the minimum energy conformation of each stereoisomer and molecular docking at Jak3 was performed in an effort to better understand each compounds selectivity and potency profiles.
PMCID: PMC2660606  PMID: 19053756
Janus Kinase 3; CP-690,550; Kinase inhibition; Chiral drugs
16.  Evaluation of Small Molecule Modulators of the Luteinizing Hormone/Choriogonadotropin and Thyroid Stimulating Hormone Receptors: Structure Activity Relationships and Selective Binding Patterns 
Journal of medicinal chemistry  2006;49(13):3888-3896.
The substituted thieno[2,3-d]pyrimidine 3 (Org 41841), a partial agonist for the luteinizing hormone/choriogonadotropin receptor (LHCGR) and the closely related thyroid-stimulating hormone receptor (TSHR), was fundamentally altered and the resulting analogues were analyzed for their potencies, efficacies and specificities at LHCGR and TSHR. Chemical modification of the parent compound combined with prior mutagenesis of TSHR provided compelling experimental evidence in support of computational models of 3 binding to TSHR and LHCGR within their transmembrane cores. Biochemical analysis of a specific modification to the chemical structure of 3 provides additional evidence of a H-bond between the ligand and a glutamate residue in transmembrane helix 3, which is conserved in both receptors. Several key interactions were surveyed to determine their respective biochemical roles in terms of both van der Waals dimensions and hydrogen bond capacity and the respective relationship to biological activity.
PMCID: PMC2543117  PMID: 16789744
Thyroid Stimulating Hormone Receptor; Luteinizing Hormone/Choriogonadotropin Receptor; Org 41841

Results 1-16 (16)