Structure-based virtual screening of small-molecule antagonists of platelet integrin αIIbβ3 that do not prime the receptor to bind ligand

Abstract

Integrin αIIbβ3 has emerged as an important therapeutic target for thrombotic vascular diseases owing to its pivotal role in mediating platelet aggregation through interaction with adhesive ligands. In the search for effective anti-thrombotic agents that can be administered orally without inducing the high-affinity ligand binding state, we recently discovered via high-throughput screening of 33,264 compounds a novel, αIIbβ3-selective inhibitor (RUC-1) of adenosine-5′-diphosphate (ADP) -induced platelet aggregation that exhibits a different chemical scaffold and mode of binding with respect to classical Arg-Gly-Asp (RGD)-mimicking αIIbβ3 antagonists. Most importantly, RUC-1 and its higher-affinity derivative, RUC-2, do not induce major conformational changes in the protein β3 subunit or prime the receptor to bind ligand. To identify additional αIIbβ3-selective chemotypes that inhibit platelet aggregation through similar mechanisms, we screened in silico over 2.5 million commercially available, 'lead-like' small molecules based on complementarity to the predicted binding mode of RUC-2 into the RUC-1-αIIbβ3 crystal structure. This first reported structure-based virtual screening application to the αIIbβ3 integrin led to the identification of 2 αIIbβ3-selective antagonists out of 4 tested, which compares favorably with the 0.003 % "hit rate" of our previous high-throughput chemical screening study. The newly identified compounds, like RUC-1 and RUC-2, showed specificity for αIIbβ3 compared to αVβ3 and did not prime the receptor to bind ligand. They thus may hold promise as αIIbβ3 antagonist therapeutic scaffolds.

Discovery of a Novel Selective Kappa-Opioid Receptor Agonist Using Crystal Structure-Based Virtual Screening

“…Similarity between these molecules and the 9,934 opioid receptor ligands that are annotated in the ChEMBL database [https://www.ebi.ac.uk/chembl/] (Table S1) was quantified using an in-house script in R language that calculates Tanimoto coefficients (T c ) to the nearest neighbors based on extended connectivity fingerprint maximum distances 4 (ECFP4) and the protocol we recently reported. 33 T c values range from 0 to 1, with the 0 value indicating maximally dissimilar compounds and 1 indicating maximally similar ones. 34 As suggested in the literature, 35 molecules are considered reasonably similar if their T c value is above 0.40.…”

Integrin αIIbβ3

“…Given the premise that targeting αIIbβ3 remains a fundamentally sound strategy, some investigators have sought to identify new αIIbβ3 antagonists, ones that might not induce conformational changes on association or dissociation from αIIbβ3 and might therefore contribute less to the bleeding and thrombocytopenia that occurs in some patients. Two possible approaches have been suggested to achieve this end: finding inhibitors that, like current antagonists, bind to the extracellular domain of the integrin but do so without promoting receptor activation 140 or finding inhibitors that prevent receptor activation by binding to the intracellular domain of αIIbβ3. 141 Both strategies are in early stages of development.…”

“…143,144 A second congener RUC-2, was ≈100-fold more potent than RUC-1 61 and did not seem to induce major conformational changes in the protein β3 subunit or prime the receptor to bind ligand. 140 RUC-2 is currently undergoing additional preclinical studies that will assess its suitability for use in patients with STEMI in the early prehospital setting. 2 …”

A Novel αIIbβ3 Antagonist from Snake Venom Prevents Thrombosis without Causing Bleeding

“…Thus, we tested the 'priming' effect of these α IIb β 3 antagonists. In this assay, the ability of agents to induce resting integrin α IIb β 3 to adopt a high-affinity ligand binding conformation was judged by measuring the intensity of fluorescence-conjugated fibrinogen ( Figure 3A) or PAC-1 ( Figure 3B) binding to platelets [25], and compared with platelets without agent administration. TFV-3, RGD-mimetic agent eptifibatide, and abciximab increased fibrinogen ( Figure 3A) or PAC-1 ( Figure 3B) binding to platelets.…”

“…The priming assay was performed as described previously, with minor modifications [25,49]. Washed platelets in HEPES-modified Tyrode's buffer were treated with eptifibatide, TFV-1, or TFV-3 for 30 min at room temperature.…”