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Integrins αVβ3 and α5β1 play critical roles in tumor survival, invasion, metastasis, and angiogenesis and are validated targets for cancer therapy and molecular imaging. Increasing evidence suggests that targeting both integrins simultaneously with antagonists is more effective in cancer therapy because of concerns about resistance and paradoxical promotion of tumor growth with use of agents highly selective for a single integrin. Engineered Arg-Gly-Asp (RGD)-containing 3.5 kDa cysteine-knot proteins (knottins 2.5F and 2.5D) are attractive drug candidates due to their exceptional structural stability and high affinity binding to certain integrins. 2.5F binds both αVβ3 and α5β1, whereas 2.5D is αVβ3-selective. To elucidate the structural basis of integrin selection, we determined the structures of 2.5F and 2.5D both as apo-proteins and in complex with αVβ3. These data, combined with MD simulations and mutational studies, revealed a critical role of two αVβ3-specific residues in the vicinity of the metal ion dependent adhesion site (MIDAS) in promoting an αVβ3-induced fit of 2.5D. In contrast, conformational selection accounted for the specificity of 2.5F to both integrins. These data provide new insights into the structural basis of integrin-ligand binding specificity, and could help in development of integrin-targeted therapeutics.

A prevailing dogma is that effective inhibition of vascular thrombosis by therapeutic targeting of platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis. The resulting serious bleeding and increased morbidity and mortality have limited use of current anti-αIIbβ3 drugs to high-risk cardiac patients. It is speculated that these adverse outcomes result from drug-induced conformational changes in αIIbβ3 but direct proof is lacking. We used structure-guided design to generate the ligand-mimetic peptide Hr10 and a modified form of the partial agonist drug Tirofiban that now act as “pure” orthosteric antagonists of αIIbβ3, i.e. they no longer induce the conformational changes in αIIbβ3 and also suppress these changes in presence of agonists. Both agents inhibited human platelet aggregation effectively but without interfering with clot retraction. When tested in a humanized mouse model of thrombosis predictive of clinical efficacy, Hr10 was as effective as the αIIbβ3 partial agonist peptide drug Eptifibatide in inhibiting arteriolar thrombosis, but in sharp contrast to Eptifibatide, Hr10 did not cause serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus offer safer alternatives for human therapy. Our structure-guided approach may also find utility in designing similar drug candidates targeting other integrins and in providing vital tools for further probing structure-activity relationships in integrins.

The transition from leukocyte rolling to firm adhesion is called arrest. β2integrins are required for neutrophil arrest1. Chemokines can trigger neutrophil arrest in vivo2and in vitro3. Resting integrins4exist in a “bent-closed” conformation, i.e., not extended (E−) and not high affinity (H−), unable to bind ligand. Electron microscopic images of isolated β2integrins in “open” and “closed” conformations5inspired the switchblade model of integrin activation from E−H−to E+H−to E+H+67. Recently8, we discovered an alternative pathway of integrin activation from E−H−to E−H+to E+H+. Spatial patterning of activated integrins is thought to be required for effective arrest, but so far only diffraction-limited localization maps of activated integrins exist8. Here, we combine superresolution microscopy with molecular modeling to identify the molecular patterns of H+E−, H−E+, and H+E+activated integrins on primary human neutrophils. At the time of neutrophil arrest, E+H+integrins form oriented (non-random) nanoclusters that contain a total of 4,625±369 E+H+β2integrin molecules.