Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function

Abstract

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6–2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Crystal structure of the kringle domain of human receptor tyrosine kinase-like orphan receptor 1 (hROR1)

“…The analysis also highlighted the unusual presence of a nonconserved lysine residue (Lys369) in hROR1-KRD in a region proximate to a hydrophobic pocket generated by the side chains of conserved residues within the two antiparallel -strands of the KRD protein fold. In the homologous plasminogen, HGF/SF and apolipoprotein A, this position is occupied by negatively charged residues (Asp or Glu), creating a strong binding site for lysine and its analogs (Hochschwender & Laursen, 1981;Va ´li & Patthy, 1980;Wu et al, 1991) that has been widely explored for its druggability (Sandmark et al, 2020;Sigurdardottir et al, 2015;Fig. 4b).…”

Lipoprotein(a): Pathophysiology, measurement, indication and treatment in cardiovascular disease. A consensus statement from the Nouvelle Société Francophone d’Athérosclérose (NSFA)

“…Similar functional analogies in the binding of Lp(a) to cellular receptors for plasminogen or to proteins with affinity for plasminogen have been demonstrated recently [89,90]. Interestingly, small-molecule inhibitors have been developed, which target the lysine-binding site of KIV 10 and are able to attenuate the pathophysiological effects of Lp(a) [91].…”

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13

“…It is possible that this was due to the low levels Lp(a) in the pooled plasma samples. In comparison to plasminogen, apo(a) contains a strong lysine binding site in KIV type 10, which shares the highest homology to plasminogen’s KIV domain, and weak lysine binding sites in KIV types 5–8 and KV 43 . The KIV type 10 site is partially occluded in apo(a) by an intramolecular interaction analogous to—but likely structurally distinct from—the closed conformation of Glu-plasminogen 44 .…”