Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.

␣v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with ArgGly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some ␣v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of ␣v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for ␣v␤3 and ␣v␤5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. IntroductionThe formation of new blood vessels from existing vasculature, or angiogenesis, is essential for successful tumor growth and for the development of metastases. Previous work has suggested that certain endothelial cell integrins, including ␣v␤3 and ␣v␤5, actively contribute to the angiogenic process. 1,2 More recently, endogenous inhibitors of angiogenesis have been shown to target these integrins. 3 Integrins are transmembrane receptors for extracellular matrix (ECM) and basement membrane proteins that are composed of 2 noncovalently associated subunits, ␣ and ␤. 4 To date, 18 ␣ and 8 ␤ subunits have been identified in mammals, and their association in various combinations leads to the formation of at least 24 receptors with distinct ligand specificity. Besides mediating stable adhesion, integrins transmit signals that regulate cell survival, growth, motility, and remodeling of their extracellular environment. [4][5][6] The integrins ␣v␤3 and ␣v␤5 bind to ECM molecules through an Arg-Gly-Asp (RGD)-binding site. Based on the concept that they are proangiogenic receptors, specific inhibitors, including blocking monoclonal antibodies, RGD peptides, and RGD peptidomimetics, have been developed and evaluated in vivo. 7 Indeed, pharmacologic agents targeted to ␣v␤3, ␣v␤5, or both, have been reported to block tumor and retinal angiogenesis. [8][9][10][11][12][13][14] Some of these angiogenesis inhibitors, including a humanized monoclonal anti␣v␤3 (Vitaxin; MedImmune, Gaithersburg, MD) and an ␣v␤3/ ␣v␤5-selective RGD-based cyclic peptide (cilengitide), have entered clinical trials. 15,16 Early experiments show that a monoclonal antibody directed against ␣v␤3 inhibits angiogenesis by inducing the apoptosis of angiogenic blood vessels. 17...

Ligand-target residence time is emerging as a key drug discovery parameter because it can reliably predict drug efficacy in vivo. Experimental approaches to binding and unbinding kinetics are nowadays available, but we still lack reliable computational tools for predicting kinetics and residence time. Most attempts have been based on brute-force molecular dynamics (MD) simulations, which are CPU-demanding and not yet particularly accurate. We recently reported a new scaled-MD-based protocol, which showed potential for residence time prediction in drug discovery. Here, we further challenged our procedure's predictive ability by applying our methodology to a series of glucokinase activators that could be useful for treating type 2 diabetes mellitus. We combined scaled MD with experimental kinetics measurements and X-ray crystallography, promptly checking the protocol's reliability by directly comparing computational predictions and experimental measures. The good agreement highlights the potential of our scaled-MD-based approach as an innovative method for computationally estimating and predicting drug residence times.