ABSTRACT
There is a grave need for safer antiplatelet therapeutics to prevent heart attack and stroke. Agents targeting the interaction of platelets with the diseased vessel wall could impact vascular disease with minimal effects on normal hemostasis. We targeted integrin alpha(2)beta(1), a collagen receptor, because its overexpression is associated with pathological clot formation whereas its absence does not cause severe bleeding. Structure-activity studies led to highly potent and selective small-molecule inhibitors. Responses of integrin alpha(2)beta(1) mutants to these compounds are consistent with a computational model of their mode of inhibition and shed light on the activation mechanism of I-domain-containing integrins. A potent compound was proven efficacious in an animal model of arterial thrombosis, which demonstrates in vivo efficacy for inhibition of this platelet receptor. These results suggest that targeting integrin alpha(2)beta(1) could be a potentially safe, effective approach to long-term therapy for cardiovascular disease.
Subject(s)
Fibrinolytic Agents/pharmacology , Integrin alpha2beta1/antagonists & inhibitors , Thrombosis/prevention & control , Allosteric Regulation , Animals , Fibrinolytic Agents/chemistry , Humans , Integrin alpha2beta1/chemistry , Mice , Mice, Inbred C57BL , Proline/analogs & derivatives , Structure-Activity Relationship , Thiazolidines/chemistry , Thiazolidines/pharmacology , beta-Alanine/analogs & derivatives , beta-Alanine/chemistry , beta-Alanine/pharmacologyABSTRACT
We herein report a group of allosteric inhibitors of integrin alpha(2)beta(1) based on an arylamide scaffold. Compound 4 showed an IC(50) of 4.80 microM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compounds are able to block collagen binding to integrin alpha(2)beta(1) on the platelet surface. Further we find that compound 4 recognizes a hydrophobic cleft on the side of the alpha(2) I-domain, suggesting an alternative targeting site for drug development.
Subject(s)
Amides/pharmacology , Collagen Type I/antagonists & inhibitors , Integrin alpha2beta1/antagonists & inhibitors , Allosteric Regulation , Amides/chemistry , Binding Sites/drug effects , Collagen Type I/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Hydrophobic and Hydrophilic Interactions , Integrin alpha2beta1/metabolism , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Structure , Protein Binding/drug effects , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Delta4-androstene-3,17-dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9alpha,11beta-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARgamma). Activation of PPARgamma is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9alpha,11beta-PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9alpha,11beta-PGF2.
Subject(s)
3-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Hydroxyprostaglandin Dehydrogenases/antagonists & inhibitors , Prostatic Diseases/enzymology , 3-Hydroxysteroid Dehydrogenases/analysis , 3-Hydroxysteroid Dehydrogenases/physiology , Aldo-Keto Reductase Family 1 Member C3 , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Dinoprost/biosynthesis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Gonadal Steroid Hormones/biosynthesis , Gonadal Steroid Hormones/metabolism , Humans , Hydroxyprostaglandin Dehydrogenases/analysis , Hydroxyprostaglandin Dehydrogenases/physiology , Male , Prostate/enzymology , Prostatic Diseases/genetics , Structure-Activity Relationship , Testosterone/biosynthesis , Transcription, GeneticABSTRACT
Tetrathiomolybdate (TM) is a potent nontoxic orally delivered copper complexing agent under development for the last several years for the treatment of Wilson's disease. It has been shown to block angiogenesis in primary and metastatic tumors. Therefore, the combination of cytotoxic radiotherapy (RT) and antiangiogenic TM could target both the existing tumor and the tumor microvasculature in a comprehensive strategy. Using a Lewis lung high metastatic (LLHM) carcinoma mouse tumor model, we demonstrate that the combination of TM and RT is more effective than either used as monotherapy. We also show that their therapeutic effects are additive, with no additional toxicity. We show that TM has no significant cytotoxicity in vitro against LLHM tumor cells, further supporting the antiangiogenic mechanism for its action.
