2,3-Diphenylpropionic acids as potent VLA-4 antagonists

The discovery and SAR of 2,3-diphenylpropionic acid derivatives as highly potent VLA-4 antagonists are described. One representative compound, 9cc has inhibited intercellular adhesion by a VCAM-1/VLA-4 interaction with an IC(50) of 1.7 nM, and has good pharmacokinetics and oral bioavailability.     

alpha1beta1 Integrin+ and regulatory Foxp3+ T cells constitute two functionally distinct human CD4+ T cell subsets oppositely modulated by TNFalpha blockade

The expression of the collagen receptor alpha(1)beta(1) integrin (VLA-1) on CD4(+) T cells is largely restricted to CCR7(-)CD45RO(+) cells that localize to inflamed tissues. Moreover, neutralizing alpha(1) integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4(+) T cells is restricted to conventional effectors. In contrast, Foxp3(+) T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4(+) T cells even upon polyclonal anti-CD3-induced activation. Because TNFalpha blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFalpha affected the balance between the proinflammatory VLA-1(+) effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFalpha blockade, inhibited the typical activation-dependent generation of CD4(+)VLA-1(+) Th1 cells. In contrast, it augmented the outgrowth of VLA-1(neg/dim)CD25(high) and Foxp3(+)CD4(+) T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFalpha blockade generated CD4(+) T cell lines enriched for VLA-1(-)Foxp3(+) Tregs. Importantly, these CD4(+) T cells displayed potent suppressive functions toward autologous CD4(+) PBL, including the suppression of the activation-dependent induction of VLA-1(+) effectors. Thus, we propose a novel mechanism by which anti-TNFalpha therapy may restore self-tolerance, by shifting the balance between VLA-1(+) effectors and Foxp3(+) Tregs, during immune activation, in favor of the latter suppressor cell population.     

Identification of unique VLA-4 antagonists from a combinatorial library

A combinatorial library of 28 pools of 180 compounds (345 diastereomers) was designed and prepared in support of the delineation of the SAR of two prototypical VLA-4 antagonists. Deconvolution of the active pools led to the identification of three novel series of VLA-4 antagonists with low nanomolar potencies.     

Sulphonamide-based small molecule VLA-4 antagonists

The discovery of a sulphonamide by-product with VLA-4 antagonistic activity led to a series of potent, small molecule VLA-4 antagonists. Synthesis, SAR and in vivo evaluation of the selected compound will be presented.     

Squaric acid derivatives as VLA-4 integrin antagonists

SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.     

Regulation of proteins in the VLA cell substrate adhesion family: influence of cell growth conditions on VLA-1, VLA-2, and VLA-3 expression

Cell quiescence resulting from culture of normal human fibroblasts in low serum (0.5%) was associated with a subsequent gradual increase in the expression of the cell-surface glycoprotein VLA-1, and a corresponding decrease in the expression of extracellular matrix adhesion receptors VLA-2 and VLA-3. Quantitation using either flow cytometry or immunoprecipitation showed that both the VLA-1/VLA-2 and VLA-1/VLA-3 ratios increased 10- to 28-fold and were still rising when cells remained quiescent for 20-30 days. Although induced by cell quiescence, changes in the levels of VLA-1, VLA-2, and VLA-3 continued to occur well after cell proliferation had stopped and thus do not directly correlate with cell cycle transition events. Despite prolonged serum deprivation resulting in elevated VLA-1/VLA-2 and VLA-1/VLA-3 ratios, growth-arrested cells remained viable and were fully capable of proliferating when restimulated. The increases in VLA-1/VLA-2 and VLA-1/VLA-3 ratios observed on quiescent cells were readily reversible, since after restimulation with 10% serum, these ratios quickly returned within 1-2 days to a level near that found on normal exponentially grown cells. Elevation of VLA-1/VLA-2 and VLA-1/VLA-3 ratios is generally associated with quiescence and is not due just to serum deprivation since density arrest of cells at confluence had similar effects on these ratios.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.

Very late Ag (VLA)-3, VLA-4, and VLA-5, belonging to the beta-1 subfamily of integrins, have been recently identified as receptors for different binding regions of fibronectin (FN). We have detected VLA-4 and VLA-5, but not VLA-3, on fresh CD3-, CD16+, CD56+ human NK cells by flow cytometry and immunochemical analyses using mAb directed against beta-1, alpha-3, alpha-4, and alpha-5 subunits. Binding assays, performed on FN-coated plates, showed that NK cells specifically adhere to FN and their binding capacity is increased by MgCl2 but not by CaCl2. Using as inhibitory probes a polyclonal antibody against the beta-1 chain of the human FN receptor, the synthetic peptide GRGDSP, which is able to inhibit cellular adhesion mediated by VLA-5, the CS1 fragment, which contains the principal adhesion site in the IIICS domain recognized by VLA-4, and functional mAb directed against alpha-4 or alpha-5 subunits, we show that both VLA-4 and VLA-5 mediate the adhesion of human NK cells to FN. The expression of these integrin receptors may be relevant for NK interaction with extracellular matrix components and other cell types.     

N-(pyrimidin-4-yl) and N-(pyridin-2-yl) phenylalanine derivatives as VLA-4 integrin antagonists

The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based VLA-4 antagonists are described.     

Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures

The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under- surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA- 3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion, our results demonstrate the expression of VLA-3 at intercellular contact sites of cultured cells supporting the concept that it may be relevant for intercellular interactions also.     

Discovery and evaluation of N-(triazin-1,3,5-yl) phenylalanine derivatives as VLA-4 integrin antagonists

SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a 1,3,5-triazine as an amide isostere are described.     

Receptor functions for the integrin VLA-3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations

The capability of the integrin VLA-3 to function as a receptor for collagen (Coll), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and ligand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carcinoma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction with Fn was often low or undetectable in cells having both VLA-3 and VLA-5, NCI-H69 cells readily attached to Fn in a VLA-3-dependent manner. Both Arg-Gly-Asp (RGD) peptide inhibition studies, and Fn fragment affinity columns suggested that VLA-3, like VLA-5, may bind to the RGD site in human Fn. However, unlike Fn, both Coll and Lm supported VLA-3-mediated adhesion that was not inhibited by RGD peptide, and was totally unaffected by the presence of VLA-5. In addition, VLA-3-mediated binding to Fn was low in the presence of Ca++, but was increased 6.6-fold with Mg++, and 30-fold in the presence of Mn++. In contrast, binding to Coll was increased only 1.2-fold with Mg++, and 1.7-fold in Mn++, as compared to the level seen with Ca++. Together, these experiments indicate that VLA-3 can bind Coll, Lm, and Fn, and also show that (a) VLA-3 can recognize both RGD-dependent and RGD-independent ligands, and (b) different VLA-3 ligands have distinctly dissimilar divalent cation sensitivities.     

Modulators of the human CCR5 receptor. Part 3: SAR of substituted 1-[3-(4-methanesulfonylphenyl)-3-phenylpropyl]-piperidinyl phenylacetamides

SAR and PK studies led to the identification of N-(1-{(3R)-3-(3,5-difluorophenyl)-3-[4-methanesulfonylphenyl] propyl}piperidin-4-yl)-N-ethyl-2-[4-methanesulfonylphenyl]acetamide as a highly potent and selective ligand for the human CCR5 chemokine receptor with good oral pharmacokinetic properties.     

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells

Background

VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions.

Methods

VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined.

Results

About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro.

Conclusions

Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration,but only a limited role in their protection by stromal cells.     

Effect of T cell activation on lymphocyte-endothelial cell adherence and the role of VLA-4 in the rat.

Lymphocyte migration from the blood is in part controlled by lymphocyte surface adhesion molecules, such as VLA-4 and LFA-1. Small lymph node lymphocytes from rats adhere poorly to rat microvascular endothelial cells (EC), while lymphoblasts from antigen-challenged lymph nodes have an enhanced adherence to EC and preferentially migrate to inflamed tissues. This lymphoblast adherence is partially inhibited by anti-VLA-4. The effects of in vitro activation of lymph node lymphocytes on lymphocyte-EC adhesion were examined. In vitro stimulation of T cells with concanavalin A or calcium ionophore and phorbol myristate acetate (PMA) for 1-4 hr caused a marked (7- to 12-fold) increase in lymphocyte adhesion to unstimulated and IFN-gamma- or LPS-treated EC. This adhesion was partially inhibited by anti-VLA-4, was not associated with increased VLA-4 expression, was partially inhibited at 4 degrees C, and was virtually eliminated at 4 degrees C with anti-VLA-4. Anti-CD3 or IL-2 stimulation of T cells also enhanced lymphocyte adhesion but required 2-3 days of culture. This adhesion was not inhibited by anti-VLA-4 and was almost totally inhibited at 4 degrees C, suggesting a primarily LFA-1-mediated adhesion. In conclusion, stimulation of T cells with Con A or calcium ionophore plus PMA caused a rapid enhancement of lymphocyte-EC adhesion mediated in part through VLA-4, while stimulation of T cells with anti-CD3 or IL-2 enhanced lymphocyte adhesion apparently independent of VLA-4.     

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.     

Expression of VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120- kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM- 1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.     

Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers

The Very Late Activation Antigen (VLA) proteins are a family of five related heterodimers, which also are part of the integrin superfamily of cell adhesion molecules. Except for the identification of VLA-5 as a fibronectin receptor structure, the functions of the VLA proteins have remained unclarified. In this paper, immunoprecipitation experiments with both anti-alpha and anti-beta subunit antibodies showed that the previously identified cell adhesion receptor for collagen, extracellular matrix receptor II (ECMRII), is equivalent to VLA-2. At the same time a previously described multispecific cell adhesion receptor for collagen, fibronectin, and laminin (ECMRI) has been shown to be identical to VLA-3. Although the mAb 12F1 and P1H5 both recognized VLA-2 (ECMRII), they appeared to define distinct epitopes on the alpha 2 subunit. On the other hand, the mAb P1B5 and J143 recognized the alpha 3 subunit of VLA-3 (ECMRI) at or near the same site. Consistent with the collagen receptor functions of VLA-2 (ECMRII) and VLA-3 (ECMRI), anti-VLA beta antiserum blocked cell attachment to collagen.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.