Probing Integrin Selectivity: Rational Design of Highly Active and Selective Ligands for the α5β1 and αvβ3 Integrin Receptor

Abstract

Rational drug design relies on an iterative procedure of initial protein-structure determination, followed by the design, chemical synthesis, and subsequent biological evaluations of specific compounds. However, there is still a large gap between known protein sequences and 3D structures. To date, the most successful theoretical approach to bridge this gap is homology modeling. It is possible to construct an approximate 3D model of the structural unknown protein if the sequence homology to the known 3D structure of the reference protein is higher than 40%. Such a homologymodeled structure is suitable for rational drug design.[1]Herein we describe the successful use of our recently published homology model of the integrin a5b1[2] to design potent (with activities up to the subnanomolar range) and selective ligands for the two highly similar integrin receptors a5b1 and avb3. Structural considerations were used to trigger potency and selectivity in both directions. These ligands could allow functional studies in vivo of the role of these two integrin subtypes and might be used as lead structures for antiangiogenic cancer therapy.Integrins constitute an important class of heterodimeric cell-adhesion receptors that are involved in many severe pathological processes, such as tumor metastasis, thrombosis, inflammation, and osteoporosis.[3] Therefore, they have been attractive therapeutic targets for several years.[4] Since Brooks et al. reported that various low-molecular-weight ligands (for example, our synthesized cyclopentapeptide cyclo(-Arg-GlyAsp-D-Phe-Val-) = c(-RGDfV-) [5a] ), which are recognized by the avb3 and avb5 integrins, block angiogenesis in response to growth factors in tumors,[5b] many selective avb3-ligands have been developed and some compounds have reached clinical trials.[7] As a result of our research, the cyclic Nmethylated pentapeptide c(-RGDf[NMe]V-), [5b] known as cilengitide, has entered phase II trials for patients with glioblastoma.Recent knock-out experiments showed, however, that genetically altered mice (lacking the av integrin) show extensive angiogenesis in some cases, whereas other mice (lacking the b3 or b5 integrins) show no significant effects, and as such, the idea that these two integrins are proangiogenic was seriously questioned. [8,9] On the other hand, the proangiogenic function of the a5b1 receptor has been clearly demonstrated [10,11] so that the a5b1 integrin moved into the focus of research. Although crystal structures of the extracellular domains of the avb3 and aIIbb3 integrins have been solved and provided a deep insight into the ligand binding, [12,13] very little detailed structural information about the a5b1 receptor itself or about ligand-receptor interactions have been obtained until now. [14] Furthermore, there are only a few small-molecule ligands known to bind a5b1, [15,16] which prompted us to focus our research on this integrin subtype. A first hint for the design of new a5b1 ligands came from our homology model of the a5b1 integrin in complex with a rece...

Zasp regulates integrin activation

“…To compare the effect of Zasp on activation of b1 and b3 integrins, we assessed the effect of Zasp expression on the activation of endogenous a5b1 integrin and stably expressed aIIbb3 integrin in CHO cells in parallel. aIIbb3-expressing CHO cells were transfected and a5b1 activation assessed with FN9-11 in the presence of the selective aIIbb3 antagonist XP-280 (Barrett et al, 1999), or the a5b1-specific antagonist 3F compound (Heckmann et al, 2007). In the same transfected CHO cell population, aIIbb3 activation was assessed with the activation-specific, ligand-mimetic, antiaIIbb3 monoclonal antibody PAC-1 (see Materials and Methods).…”

“…To block background FN9-11 binding to aIIbb3, XP280 was added to cells incubated with FN9-11 in the presence or absence of EDTA. Alternatively, an a5b1-specific inhibitor, 3F, kindly provided by Dr Horst Kessler (Heckmann et al, 2007), was used to specifically block FN9-11-binding to a5b1. Total a5b1 integrin expression levels were measured in parallel by staining with PB1 (Brown and Juliano, 1985).…”

High‐Affinity “Click” RGD Peptidomimetics as Radiolabeled Probes for Imaging αvβ3 Integrin

“…phenyl, isoxazole, 1,2,3-triazole rings and hydrazide bonds) in the flexible peptide sequence. [9][10][11][12][13] In fact, the reduction of conformational freedom towards a correct bioactive orientation favorably affects both activity and selectivity. From several studies focusing on the development of non-peptide RGD mimics, L-2,3-diaminopropanoic acid emerged as an optimal replacement of aspartic acid.…”

“…[10,11] To improve the pharmacological features of RGD peptides, a number of RGD peptidomimetics have been developed as therapeutic agents. [14] However, relatively few studies have investigated the use of these molecules as radiotracers.…”

Targeted-TERS detection of integrin receptors on human cancer cells

“…The observed vibrational bands can be assigned to amino acids found in the RGD binding sites of α5β1 and αvβ3 integrins [46,47] . SERS spectra exhibit distinct differences in Raman bands (Figure 1b), arising from differences in ligand binding sites between α5β1 and αvβ3 receptors [48,49] . The acquired SERS spectra were analyzed with multivariate curve resolution (MCR), a chemometric method that analyzes the variance in the spectra and generates pure components associated with the spectral composition.…”