Therapeutic antagonists and the conformational regulation of the beta2 integrins

The beta2 integrins are validated therapeutic targets for inflammatory disorders. Two distinct mechanistic classes of small molecule inhibitors, termed alpha I allosteric and alpha/beta I-like allosteric antagonist, have recently been developed. The alpha I allosteric antagonists bind underneath the C-terminal helix of the I domain and stabilize the I domain in the inactive closed conformation. By contrast, the alpha/beta I-like allosteric antagonists bind to the beta2 I-like domain MIDAS and disrupt conformational signal transmission between the I and the I-like domain, leaving the I domain in a default inactive form. Furthermore, the two classes of the antagonists have opposite effects on integrin conformation; the alpha I allosteric antagonists stabilize the bent conformation, whereas the alpha/beta I-like allosteric antagonists induce the extended conformation with inactive I domain. The small molecule antagonists to the beta2 integrin highlight the importance of the structural linkages within and between integrin domains for transmission of the conformational signals and regulation of the overall conformation.     

Small molecule antagonists of alpha4 integrins: novel drugs for asthma

The alpha4 integrins, alpha4beta1 (VLA-4) and alpha4beta7, are heterodimeric cell-surface proteins expressed on leukocytes involved in both cell-cell and cell-matrix interactions. These two integrins are key regulators of physiologic and pathologic responses in inflammation and autoimmune disease. In particular, their central role in animal models of allergic lung disease has been well documented, and suggests a key role in human asthma. In addition, integrins are proven valid targets for small molecule drugs, following the development of both parenteral and orally active antagonists of platelet integrin alphaIIbbeta3, and more recently, integrin alphaVbeta3. Therefore, there is a significant drive to develop small molecule inhibitors of alpha4 integrins for autoimmune diseases in general, and asthma in particular. The biology of alpha4 integrins in asthma has been recently reviewed, as has early work on the chemistry of alpha4 integrin antagonists. Here we summarise the recent rapid advances in this arena, particularly in the chemistry of alpha4 integrin small molecule antagonists, with emphasis on asthma as a clinical target.     

Dual effect of alpha-adrenoceptor antagonists in rat isolated vas deferens.

1 In rat isolated vas deferens, the isotonic contractile responses to low doses of noradrenaline or adrenaline were antagonized, and those to high doses were potentiated, by yohimbine, piperoxan, phentolamine and tolazoline. Effects due to intermediate doses were not affected, or were potentiated within about 30 min, following an initial inhibition. 2 The alpha-adrenoceptor blockers thus caused a shift to the right and an increase of the maximum height of log dose-response curves of alpha-adrenoceptor stimulants. For a given dose of antagonist, the onset was slower for the potentiating than for the blocking effect. 3 The shift to the right induced by piperoxan and yohimbine on dose-response curves of noradrenaline and adrenaline was analysed with the Schild plot, and the slopes obtained, around 0.3, were lower than expected from receptor theory. When cocaine was used to block neuronal uptake, the slopes were close to 1.0. 4 The increase in maximum response to noradrenaline and adrenaline induced by alpha-adrenoceptor blockers was dependent on the time of incubation, on the dose of antagonist, and on the initial height of responses to the agonist. A less pronounced potentiation was also obtained when acetylcholine was used as agonist. 5 The findings are explained in terms of receptor theory as being due to a dual effect of alpha-adrenoceptor antagonists; competitive antagonism proper, which may be disclosed after blockade of neuronal uptake, and an interaction at a different locus, which results in potentiation of the effects of noradrenaline and adrenaline.     

Inhibitors of integrins

The inhibition of integrins--cell surface receptors with a crucial role in angiogenesis, tumour cell survival, invasion and metastases--has centred on the alpha(v)beta3 integrin. Work has culminated in two antagonists that are in clinical trials as cancer therapeutics. Other integrins appear to be candidate targets in the light of gene knockout studies. Surprisingly, genetic alpha(v)beta3 ablation did not confirm the pertinence of the use of alpha(v)beta3 antagonists. However, these apparent discrepancies could be explained by the new finding that this integrin has a role as a cell survival sensor, limiting rather than promoting angiogenesis. Accumulating data on the role of integrins and the mechanism of action of pharmacological antagonists will help to develop and apply an efficient anti-integrin therapy in cancer.     

Dual mode of action of dihydropyridine calcium antagonists: a role for nitric oxide.

Dihydropyridine calcium antagonists have been used for many years in the treatment of angina pectoris and hypertension. According to the common view, their mechanism of action is based on an inhibition of the smooth muscle L-type calcium current, thus decreasing intracellular calcium concentration and inducing smooth muscular relaxation. However, in recent years evidence has accumulated that besides the smooth muscle effects of these agents, their effect on the endothelium must also to be taken into account. It was shown that dihydropyridines can induce the release of nitric oxide (NO) from the vascular endothelium of various vessels and in different species. This was first shown by Günther and colleagues by assaying the methaemoglobin formation in the presence of intact endothelium (in porcine coronary arteries) with and without treatment with nitrendipine. These findings were later confirmed by direct measurement of NO or of nitrite production. In addition, in several preparations, including micro- and macrovasculature, the sensitivity of the vasorelaxing effect of the dihydropyridines to inhibitors of NO-synthase, such as L-N(G)-nitroarginine (LNNA) or L-N-nitro-arginine-methyl-ester (L-NAME), has been shown. With these studies it became evident that the NO-releasing effect was not unique to nitrendipine but was a group phenomenon shared by the dihydropyridines and several nondihydropyridine calcium antagonists. In addition to their action on vascular endothelium, NO release by nifedipine has also been detected in platelets. There are also studies showing long term effects of calcium antagonists involving NO release. Regarding the underlying mechanism of NO release, nitrendipine was shown, not to decrease but to increase intracellular Ca2+ in cultured endothelial cells. This increase was sensitive to both Ca2+-free extracellular superfusion and to gadolinium, a lanthanide known to inhibit shear-stress activated cation channels. This increase in intracellular calcium can activate endothelial NO-synthase, thus inducing the release of NO. These findings on a dual mode of action, i.e. the direct relaxing effect by inhibition of the smooth muscle L-type calcium current and indirect relaxing effect by release of NO from vascular endothelium may help to understand the beneficial antihypertensive effects of the dihydropyridine calcium antagonists and the preferential effect of certain drugs in certain vascular regions (resistance versus conductive vessels). In addition, NO release from both vascular endothelium and platelets may contribute to the antiatherosclerotic and antithrombotic effects described for certain dihydropyridines.     

Targeting integrins in hepatocellular carcinoma

INTRODUCTION: Integrins, which are heterodimeric membrane glycoproteins, consist of a family of cell-surface receptors mediating cell-matrix and cell-cell adhesion. Analysis of tumor-associated integrins has revealed an important relationship between integrins and tumor development, bringing new insights into integrin-based cancer therapies. Hepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide and integrins appeal to be a novel group of potential therapeutic targets for HCC. AREAS COVERED: This review summarizes the current knowledge of integrins involved in HCC and the potential of integrin-targeted drugs in HCC therapy. A brief introduction on the structure, biological function and regulatory mechanism of integrins is given. The distinct expression patterns and biological functions of HCC-associated integrins are described. Finally, the current situation of integrin-based therapies in HCC and other tumor types are extensively discussed in the light of their implications in preclinical and clinical trials. EXPERT OPINION: To date, increasing numbers of integrin-targeted drugs are undergoing development and they exhibit diverse effects in cancer clinical trials. Tumor heterogeneity should be emphasized in developing effective integrin-targeted drugs specific for HCC. A better understanding of how integrins cooperatively function in HCC will assist in designing more successful integrin-targeted therapeutic drugs and corresponding approaches.     

Targeting integrins in hepatocellular carcinoma

Introduction: Integrins, which are heterodimeric membrane glycoproteins, consist of a family of cell-surface receptors mediating cell–matrix and cell–cell adhesion. Analysis of tumor-associated integrins has revealed an important relationship between integrins and tumor development, bringing new insights into integrin-based cancer therapies. Hepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide and integrins appeal to be a novel group of potential therapeutic targets for HCC.

Areas covered: This review summarizes the current knowledge of integrins involved in HCC and the potential of integrin-targeted drugs in HCC therapy. A brief introduction on the structure, biological function and regulatory mechanism of integrins is given. The distinct expression patterns and biological functions of HCC-associated integrins are described. Finally, the current situation of integrin-based therapies in HCC and other tumor types are extensively discussed in the light of their implications in preclinical and clinical trials.

Expert opinion: To date, increasing numbers of integrin-targeted drugs are undergoing development and they exhibit diverse effects in cancer clinical trials. Tumor heterogeneity should be emphasized in developing effective integrin-targeted drugs specific for HCC. A better understanding of how integrins cooperatively function in HCC will assist in designing more successful integrin-targeted therapeutic drugs and corresponding approaches.     

Collagen-binding integrins as pharmaceutical targets

In recent years, our understanding of the molecular interaction of collagens with their cognate integrin receptors has remarkably improved. Structural elucidations of both the integrin and the collagenous triple helix have contributed to this achievement. The structures of an entire integrin ectodomain and of an A-domain, which is unique to the integrin alpha subunits of collagen-binding and leukocyte integrins, have been resolved crystallographically. Furthermore, a complex of such an integrin alpha subunit A-domain with its collagenous binding partner has revealed their interaction on the molecular level and gave first evidence in the conformational alterations which may convey the signal of ligand occupancy through the integrin into the cells. In parallel, the tissue distribution and biological functions of collagen-binding integrins have been characterised. Nowadays, the contribution of distinct integrins to different physiological and pathological processes is known. Among the best studied examples is the collagen-induced platelet activation and aggregation, in which alpha2beta1 integrin is involved. Together with alpha1beta1 integrin, it also plays a role in inflammatory processes. To manipulate processes which are mediated by collagen-binding integrins, compounds are developed which mimic the collagen ligand. Not only the structural information of the integrin:collagen-interaction but also improvements in the chemical synthesis of a collagenous triple helix facilitate the development of agonists and antagonists of collagen-binding integrins. Furthermore, another impact in this search comes from the discovery of high-affinity inhibitors from venoms, which lack a collagenous triple-helix.     

Sodium dodecyl sulfate-stable complexes of echistatin and RGD-dependent integrins: a novel approach to study integrins

This study shows that disintegrins, echistatin as a model, can be used as a radiolabeled probe to simultaneously detect the presence of individual RGD-dependent integrins on cardiac fibroblasts. Binding of (125)I-echistatin to fibroblasts was proportional to cell number, time dependent, reversible, saturable, specific, and membrane bound. SDS-polyacrylamide gel electrophoresis and autoradiograms revealed that (125)I-echistatin was associated with three radioactive protein bands of 180, 210, and 220 kDa that were identified by RGD affinity chromatography, immunoblotting, and immunoneutralization as alpha(v)beta(3), alpha(3)beta(1)/alpha(5)beta(1)/alpha(v)beta(1), and alpha(8)beta(1) heterodimeric integrins, respectively. These results suggest that echistatin binds to RGD-dependent integrins, forming SDS-stable complexes in the absence of chemical cross-linkers, reducing conditions and heating. As assessed by radioligand-binding filtration, disintegrins displayed binding characteristics with an IC(50) ranging from 0.044 to 1.1 nM, but with slope factors lower than 1, indicating the presence of several binding sites. Resolved by SDS-polyacrylamide gel electrophoresis to reveal echistatin-integrin complexes, disintegrins and RGD peptides displayed different binding affinities to individual RGD-dependent integrins present on cardiac fibroblasts. Elegantin and flavostatin demonstrated the highest affinity toward integrins, whereas flavoridin and acPenRGDC had a greater specificity toward alpha(v)beta(3)-integrin. In summary, echistatin forms SDS-stable complexes with RGD-dependent integrins. This model offers a novel way to visualize RGD-dependent integrins, to investigate their activation state, and to determine the integrin specificity of RGD peptides.     

Drugs targeting integrins for cancer therapy

Background: Integrins are cell adhesion receptors involved in development, angiogenesis, blood clotting, inflammation and cancer. Abnormal integrin expression is a hallmark of cancer and angiogenic endothelial cells. Integrin-targeted therapy is, therefore, considered a promising novel treatment approach in oncology. Objective: We describe the biological background making integrins an attractive therapeutic target as well as the effects of integrin-targeted therapies in preclinical and clinical settings. Methods: A literature search in integrin-targeted therapy was conducted, focusing on α_vβ₃, α_vβ₅, α₅β₁ and α₄β₁ integrin ligands as well as in vivo models and clinical trials. Results/conclusion: Blocking certain integrins can inhibit tumor growth and integrin ligands can be used to target cytotoxic agents to cancer tissue. Clinical trials using integrin inhibitors have yielded variable results and continuing studies evaluate their role as monotherapy or in combination with chemo- or radiotherapy.     

Imaging of integrins as biomarkers for tumor angiogenesis

The integrin family plays important roles during tumor angiogenesis, the formation of new blood vessels from pre-existing vasculature. Traditional structural and functional imaging techniques are not sufficient for early lesion detection, patient stratification, or monitoring the therapeutic efficacy against cancer. Molecular imaging, the visualization, characterization and measurement of biological processes at the molecular and cellular levels in humans and other living systems, can fulfill these goals. In this review article, we will summarize the current state-of-the-art of imaging integrin (alpha(2)beta(1), alpha(3)beta(1), alpha(4)beta(1), alpha(v)beta(3), and alpha(v)beta(6)) expression using either single molecular imaging modality (magnetic resonance imaging, ultrasound, optical, single photon emission computed tomography, and positron emission tomography) or a combination of different modalities. For clinical translation, radionuclide-based imaging will have broad potential applications in cancer patients and the currently available clinical data (exclusively on integrin alpha(v)beta(3) so far) will be discussed in detail. The design, optimization, and characterization of imaging agents targeting integrins will be presented and areas needing extensive future research effort will be discussed. In the new era of personalized medicine, fast clinical translation and incorporation of integrin imaging into anti-cancer clinical trials will be critical for the maximum benefit of cancer patients.     

Targeting integrins for the control of tumour angiogenesis

The crucial role of cell extracellular matrix communication in angiogenesis is well established; thus, it is not surprising that integrins have gained considerable attention as targets for the treatment of neovascular disease. Given the diversity of ligands and complexity of integrin signalling, a new appreciation for the divergent roles of integrins in angiogenesis is emerging. It is becoming clear that integrins regulate angiogenesis in both a positive and negative manner. New studies have provided a better understanding of integrin structure as it relates to ligand binding and signalling. This new insight has opened exciting possibilities for the design of novel inhibitors for clinical applications. In this review, studies concerning the cooperative interactions between integrins and regulatory molecules and possible new strategies for controlling angiogenesis will be discussed.     

Targeting integrins for the control of tumour angiogenesis

The crucial role of cell extracellular matrix communication in angiogenesis is well established; thus, it is not surprising that integrins have gained considerable attention as targets for the treatment of neovascular disease. Given the diversity of ligands and complexity of integrin signalling, a new appreciation for the divergent roles of integrins in angiogenesis is emerging. It is becoming clear that integrins regulate angiogenesis in both a positive and negative manner. New studies have provided a better understanding of integrin structure as it relates to ligand binding and signalling. This new insight has opened exciting possibilities for the design of novel inhibitors for clinical applications. In this review, studies concerning the cooperative interactions between integrins and regulatory molecules and possible new strategies for controlling angiogenesis will be discussed.     

Interactions of calmodulin antagonists with calcium antagonists binding sites

Calmodulin antagonists have calcium entry blocking properties. In order to quantitatively investigate the interactions of these drugs with calcium channels, their effect on [³H]nitrendipine and [³H]d-cis-diltiazem binding to rat cerebral cortex membrane preparations was compared to their inhibitory effect on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The potency of most antagonists to inhibit [³H]nitrendipine binding was correlated with their calmodulin inhibitory potency. However, bepridil (K_0.5 = 280 nM), chlorpromazine (K_0.5 = 3 μM), triflupromazine (K_0.5 = 1.5 μM), imipramine (K_0.5 = 3 μM) and propranolol (K_0.5 = 14 μM) were much more active on [³H]d-cis-diltiazem binding than on either [³H]nitrendipine binding or calmodulin, suggesting that these compounds bind to higher affinity sites on the calcium antagonist target proitein. Moreover, the potencies of these compounds to compete with [³H]d-cis-diltiazem and to inhibit calcium-induced contractions in depolarized smooth muscle were correlated (R = 0.76, p < 0.02). These data suggest that love concentrations of these hydrophobic drugs which have calcium and calmodulin antagonistic properties inhibit smooth muscle contraction through calcium entry blockade, not calmodulin antagonism.     

NMDA receptor antagonists and glycine site NMDA antagonists

Extracellular concentrations of excitatory amino acids increase substantially within cerebral tissue beds exposed to ischaemic conditions. This leads to excessive stimulation of N-methyl-D-aspartate (NMDA) receptors, a major cerebral excitatory neurotransmitter receptor that likely plays a critical role in the propagation of ischaemic injury in neurons. Pharmacological blockade of these receptors has proven to be an effective neuroprotective therapy by a number of animal models of central nervous system ischaemia. Clinical trials of these drugs were begun with high expectations for successful therapy of human stroke. These putative neuroprotective drugs included competitive or non-competitive inhibitors of the NMDA receptor itself, as well as inhibitors of a co-modulatory glycine site. Thus far, all clinical trials of NMDA antagonists have been unsuccessful in establishing benefit for human stroke.     

Dual angiotensin II and endothelin A receptor antagonists: synthesis of 2'-substituted N-3-isoxazolyl biphenylsulfonamides with improved potency and pharmacokinetics

In a previous report we demonstrated that merging together key structural elements present in an AT(1) receptor antagonist (1, irbesartan) with key structural elements in a biphenylsulfonamide ET(A) receptor antagonist (2) followed by additional optimization provided compound 3 as a dual-action receptor antagonist (DARA), which potently blocked both AT(1) and ET(A) receptors. Described herein are our efforts directed toward improving both the pharmacokinetic profile as well as the AT(1) and ET(A) receptor potency of 3. Our efforts centered on modifying the 2'-side chain of 3 and examining the isoxazolylsulfonamide moiety in 3. This effort resulted in the discovery of 7 as a highly potent second-generation DARA. Compound 7 also showed substantially improved pharmacokinetic properties compared to 3. In rats, DARA 7 reduced blood pressure elevations caused by intravenous infusion of Ang II or big ET-1 to a greater extent and with longer duration than DARA 3 or AT(1) or ET(A) receptor antagonists alone. Compound 7 clearly demonstrated superiority over irbesartan (an AT(1) receptor antagonist) in the normal SHR model of hypertension in a dose-dependent manner, demonstrating the synergy of AT(1) and ET(A) receptor blockade in a single molecule.