Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering

Abstract

We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin alpha5beta1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the alpha4beta1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both alpha5beta1 and alpha4beta1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (a disintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.

Integrin αvβ3‐targeted cancer therapy

“…Since then, many more members of this protein family have been discovered and isolated, such as kistrin [Dennis et al, 1990], bitistatin [Shebuski et al, 1989], barbourin [Scarborough et al, 1991], echistatin [Gan et al, 1988], and contortrostatin [Swenson et al, 2004]. Because of their high integrin binding affinities and their inhibition efficacy, several disintegrins and other integrin inhibiting snake venom components have been evaluated in pre-clinical tests for their capacity to inhibit tumor growth and progression both in vitro and in vivo [Calvete et al, 2003;McLane et al, 2001;Zhou et al, 2004]. However, as proteins they would also elicit an immunological reaction in the patient.…”

Concerted Motions of the Integrin-binding Loop and the C-terminal Tail of the Non-RGD Disintegrin Obtustatin

“…Disintegrins are divided into five different groups (10). The first group includes short disintegrins composed of 49 -51 residues and four disulfide bonds.…”

Differences in binding of 99mTc-disintegrins to integrin αvβ3 on tumor and vascular cells

“…Among disintegrins tested in this study, 99m Tc-VLO4 had the highest binding to HUVEC and was among the top three in binding to cancer cells. VLO4 recognizes α5β1 > αIIbβ3 > α4β1 on CHO cells transfected with defined integrins, but does not bind to α1β1, α2β1 or α6β1 [22]. Selectivity of VLO4 for αvβ3 has not been reported.…”

“…The homodimeric disintegrin VLO4 was recently reported as having potent binding to α5β1 [22], with higher affinity than kistrin or flavoridin (see Table 3). Among disintegrins tested in this study, 99m Tc-VLO4 had the highest binding to HUVEC and was among the top three in binding to cancer cells.…”

“…Other disintegrins were produced by reversed-phase (RP) HPLC purification from freeze-dried natural snake venom (Miami Serpentarium, Punta Gorda, FL): kistrin (from Calloselasma rhodostoma venom) [19]; echistatin (from Echis carinatus) [20]; flavoridin (from Trimeresurus flavoviridis) [21]; and VLO4 (homodimer from Vipera lebetina obtusa) [22]. The active component was identified by its bioactivity (e.g., ability to inhibit platelet aggregation in human platelet-rich plasma, or ability to inhibit adhesion of cultured cells [22]). The active material was purified to a single peak on RP-HPLC.…”