Studies on selectin blocker. 9. SARs of non-sugar selectin blocker against E-, P-, L-selectin bindings.

As a part of study of selectin blockers, we have already reported that a non-sugar selectin antagonist (3) was successfully discovered using a computational screening (Hiramatsu, Y.; Tsukida, T.; Nakai, Y.; Inoue, Y.; Kondo, H. J. Med. Chem. 2000, 43, 1476). To investigate the SARs of compound 3 against E-, P-, and L-selectins, we synthesized the derivatives of compound 3 and evaluated their inhibitory activities toward selectin bindings. The structural diversity of compound 3 contained the following: (1) a modification of the spacer unit (4--7), (2) a modification of the tail unit (8--11), (3) a modification of the head unit (12--18). As a result, it was found that a non-sugar based selectin blocker (3) could be a potential lead compound for E-, P-, and L-selectin blockers and some of the derivatives showed broad and/or selective inhibitory activities toward the E-, P-, and L-selectins. In addition, it was found that the experimental evidence well supported that the computational screening using 3D-pharmacophore model could be useful methodology to find out a new lead for the several type of selectin blockers, which included a broad and/or a selective inhibitor.     

Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins

Leukocyte interactions with vascular endothelium at sites of inflammation can be dynamically regulated by activation-dependent adhesion molecules. Current models, primarily based on studies with polymorphonuclear leukocytes, suggest the involvement of multiple members of the selectin, integrin, and immunoglobulin gene families, sequentially, in the process of initial attachment (rolling), stable adhesion (arrest), spreading and ultimate diapedesis. In the current study, IL-4-activated human umbilical vein endothelium, which selectively expresses VCAM-1 and an L-selectin ligand but not E- selectin, and appropriate function blocking monoclonal antibodies, were used to study monocyte-endothelial interactions in an in vitro model that mimics microcirculatory flow conditions. In this system, L- selectin mediates monocyte rolling and also facilitates alpha 4 beta 1- integrin-dependent arrest, whereas beta 2-integrins are required for spreading of firmly attached monocytes on the endothelial cell surface but not their arrest. These findings provide the first in vitro evidence for human monocyte rolling on cytokine-activated endothelium, and suggest a sequential requirement for both beta 1- and beta 2- integrin-dependent adhesive mechanisms in monocyte-endothelial interactions.     

Selectins and glycosyltransferases in leukocyte rolling in vivo

Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified - P-, E- and L-selectin - which recognize and bind to crucial carbohydrate determinants on selectin ligands. Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo conditions. Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level. However, high endothelial venule-expressed selectin ligands were instrumental in uncovering the crucial role of post-translational modifications for selectin ligand activity. Several glycosyltransferases, such as core 2 beta1,6-N-acetylglucosaminyltransferase-I, beta1,4-galactosyltransferases, alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins. In addition, modifications, such as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands.     

Potential selectin L ligands involved in selective recruitment of peripheral blood CD16(-) natural killer cells into human endometrium

Unique CD16(-) natural killer (NK) cells appear in the human cycling endometrium and acutely increase in number after ovulation. Selective recruitment from peripheral blood (PB) CD16(-) NK cells is a potential mechanism for the postovulatory increase of these NK cells. The interaction between selectin L, an adhesion molecule playing a critical role in leukocyte extravasation, and its ligands may be involved in this phenomenon. We investigated the menstrual cycle-dependent fluctuation of selectin L expression on PB CD16(-) NK cells and selectin L ligand expression in the human endometrial endothelium. The expression of selectin L on PB CD16(-) NK cells was constantly high throughout the menstrual cycle compared with other PB CD16(+) NK cells and non-NK lymphocytes. Among eight selectin L ligands examined, podocalyxin-like, mucosal addressin cell adhesion molecule-1 (MADCAM1) and chondroitin sulfate proteoglycan 2 (CSPG2) were localized in the endometrial endothelium. Semiquantitative score of immunostaining intensity in the endometrial endothelium for MADCAM1 was highest in the late secretory phase, whereas that for CSPG2 peaked throughout the secretory phase. There was a strong positive correlation between the number of endometrial NK cells and the semiquantitative score for CSPG2. Three active isoforms of CSPG2 mRNA were detected in the human endometrium. These findings support the idea that the interaction between selectin L and selectin L ligands functions in the postovulatory selective recruitment of PB CD16(-) NK cells into the human endometrium.     

Design, synthesis, and studies of small molecule STAT3 inhibitors

A series of small molecule STAT3 inhibitors originally derived from our lead compound STA 21 were synthesized and evaluated. The most potent compound in this series, compound 1, exhibited the same anti-proliferative activities as STA 21 against prostate cancer cell lines that express constitutively active STAT3. Molecular docking showed compound 1 bound to the STAT3beta SH2 domain in a similar manner as STA 21.     

Delivery of apoptotic signal to rolling cancer cells: A novel biomimetic technique using immobilized TRAIL and E‐selectin

The survival rate for patients with metastases versus localized cancer is dramatically reduced, with most deaths being associated with the formation of secondary tumors. Circulating cancer cells interact with the endothelial lining of the vasculature via a series of adhesive interactions that facilitate tethering and firm adhesion of cancer cells in the initial steps of metastasis. TNF‐related apoptosis‐inducing ligand (TRAIL) holds promise as a tumor‐specific cancer therapeutic, by inducing a death signal by apoptosis via the caspase pathway. In this study, we exploit this phenomenon to deliver a receptor‐mediated apoptosis signal to leukemic cells adhesively rolling along a TRAIL and selectin‐bearing surface. Results show that cancer cells exhibit selectin‐mediated rolling in capillary flow chambers, and that the rolling velocities can be controlled by varying the selectin and selectin surface density and the applied shear stress. It was determined that a 1 h rolling exposure to a functionalized TRAIL and E‐selectin surface was sufficient to kill 30% of captured cells compared to static conditions in which 4 h exposure was necessary to kill 30% of the cells. Thus, we conclude that rolling delivery is more effective than static exposure to a TRAIL immobilized surface. We have also verified that there is no significant effect of TRAIL on hematopoietic stem cells and other normal blood cells. This represents the first demonstration of a novel biomimetic method to capture metastatic cells from circulation and deliver an apoptotic signal. Biotechnol. Bioeng. 2009;102: 1692–1702. © 2008 Wiley Periodicals, Inc.     

Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow

Memory T lymphocytes extravasate at sites of inflammation, but the mechanisms employed by these cells to initiate contact and tethering with endothelium are incompletely understood. An important part of leukocyte extravasation is the initiation of rolling adhesions on endothelial selectins; such events have been studied in monocytes and neutrophils but not lymphocytes. In this study, the potential of T lymphocytes to adhere and roll on endothelial selectins in vitro was investigated. We demonstrate that T cells can form tethers and rolling adhesions on P selectin and E selectin under physiologic flow conditions. Tethering and rolling on P selectin was independent of cell- surface cutaneous lymphocyte antigen (CLA) expression, which correlated strictly with the capacity of T cells to form rolling adhesions under flow on E selectin. T cell tethering to P selectin was abolished by selective removal of cell surface sialomucins by a P. haemolytica O- glycoprotease, while cutaneous lymphocyte antigen expression was unaffected. A sialomucin molecule identical or closely related to P selectin glycoprotein ligand-1 (PSGL-1), the major P selectin ligand on neutrophils and HL-60 cells, appears to be a major T cell ligand for P selectin. P selectin glycoprotein ligand-1 does not appear to support T cell rolling on E selectin. In turn, E selectin ligands do not appear to be associated with sialomucins. These data demonstrate the presence of structurally distinct ligands for P or E selectins on T cells, provide evidence that both ligands can be coexpressed on a single T cell, and mediate tethering and rolling on the respective selectins in a mutually exclusive fashion.     

Structure-activity relationships of novel, highly potent, selective, and orally active CCR1 antagonists

Design and synthesis of a series of 3-amino-4-(2-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy)phenylamino)cyclobutenedione derivatives as novel CCR1 antagonists are described. Structure-activity relationship studies led to the identification of compound 22, which demonstrated potent binding activity, functional antagonism of CCR1 as well as good species cross-reactivity. In addition, compound 22 also showed desirable pharmacokinetic profiles and was selected for in vivo studies in the mouse collagen-induced arthritis model.     

Use of core modification in the discovery of CC214-2, an orally available,selective inhibitor of mTOR kinase

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.     

E‐Selectin Genotypes and Risk of Type 2 Diabetes in Women

Endothelial dysfunction increases risk for type 2 diabetes. We examined whether variation in the gene for E‐selectin (SELE), a biomarker of endothelial dysfunction, was associated with levels of E‐selectin or diabetes quantitative traits (including fasting levels of insulin and hemoglobin A_1c) in 719 nondiabetic participants of the Nurses’ Health Study or with risk of diabetes in 602 incident (over 10 years of follow‐up) cases and 655 control women matched for age, race, and fasting status. Variation in three single nucleotide polymorphisms previously associated with cardiovascular disease risk and having effects on E‐selectin function, S128R, G98T, and L554F, was not significantly (p > 0.05) associated with levels of E‐selectin or diabetes quantitative traits, or with risk of incident diabetes in the primary analysis. Among women with low levels of subclinical inflammation (C‐reactive protein levels below the population median), S128R R allele carriers had a diabetes risk factor‐adjusted relative risk of incident diabetes of 1.71 (95% confidence interval, 1.04 to 2.81) relative to those with the SS genotype. Apart from an association in this subgroup, we conclude that the E‐selectin variants we examined are not important genetic risk factors for type 2 diabetes in women.     

P-Selectin-mediated acute inflammation can be blocked by chemically modified heparin, RO-heparin

Selectins are carbohydrate-binding cell adhesion molecules that play a major role in the initiation of inflammatory responses. Heparin can bind to P-selectin, and its anti-inflammatory property is mainly due to inhibition of P-selectin. However, the strong anticoagulant activity of heparin limits its clinical use. We prepared periodate-oxidized, borohydride-reduced heparin (RO-heparin) by chemical modification and tested its anticoagulant and anti-inflammatory activities. Activated partial thromboplastin time (aPTT) assays showed that, compared with heparin, RO-heparin had greatly reduced anticoagulant activity. Intravenous administration of this compound led to reduction in the peritoneal infiltration of neutrophils in a mouse acute inflammation model. In vitro cell adhesion experiments demonstrated that the effect of RO-heparin on inflammatory responses was mainly due to inhibiting the interaction of P-selectin with its ligands. These results indicate that RO-heparin may be a safer treatment for inflammation than heparin, especially when selectin is targeted.     

Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor

We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)]. The combination of P- and L-selectin antibodies suppressed CFVs in 60% of treated dogs (P = NS). In contrast, systemic selectin blockade by intravenous infusion or local adventitial application of MBPA markedly reduced CFVs and, in addition, reduced myocardial myeloperoxidase (MPO) activity. We conclude that inhibition of L-, P-, and E-selectin binding by a small-molecular-weight, noncarbohydrate compound markedly reduces arterial thrombosis, whereas systemic administration of antibodies to L- and P-selectin fail to reproduce this antithrombotic effect. These results underscore the role of selectins in the pathogenesis of arterial thrombosis under high shear stress and suggest that inhibition of P- and L- selectin may not suffice to prevent thrombus formation in this model. The role of E-selectin in thrombus formation in this model awaits further testing.     

A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922-1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539-542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant (k(off)) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k(off) values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s(-1) for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 A for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.     

Design, synthesis, and biological activity of thiazole derivatives as novel influenza neuraminidase inhibitors

A series of novel influenza neuraminidase (NA) inhibitors based on thiazole core were synthesized and evaluated for their ability to inhibit NA of influenza A virus (H(3)N(2)). All compounds were synthesized in good yields starting from commercially available 2-amino-4-thiazole-acetic ester using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A NA. The most potent compound of this series is compound 4d (IC(50)?=?3.43 μM), which is about 20-fold less potent than oseltamivir, and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazole ring.     

Potent, elective human beta3 adrenergic receptor agonists containing a substituted indoline-5-sulfonamide pharmacophore.

A series of compounds possessing an N-substituted indoline-5-sulfonamide pharmacophore was prepared and evaluated for their human beta3 adrenergic receptor agonist activity. The SAR of a wide range of urea and heterocyclic substituents is discussed. 4-Octyl thiazole compound 8c was the most potent and selective compound in the series, with 2800-fold selectivity over beta1 binding and 1400-fold selectivity over beta2 binding.     

PI3K is involved in L‐selectin‐ and PSGL‐1‐mediated neutrophil rolling on E‐selectin via F‐actin redistribution and assembly

L‐selectin and P‐selectin glycoprotein ligand‐1 (PSGL‐1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F‐actin changes mediated by L‐selectin and PSGL‐1 during neutrophil rolling on E‐selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L‐selectin and PSGL‐1 with E‐selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine‐phosphorylated, depending on PI3K. Furthermore, the F‐actin redistribution and assembly mediated by ligation with E‐selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E‐selectin. However, Syk/Zap70, the well‐known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F‐actin‐based cytoskeleton changes during neutrophil rolling on E‐selectin, which may consequently regulate the rolling event. J. Cell. Biochem. 110: 910–919, 2010. © 2010 Wiley‐Liss, Inc.     

Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.     

Activin,a Grape Seed-derived Proanthocyanidin Extract,Reduces Plasma Levels of Oxidative Stress and Adhesion Molecules (ICAM-1, VCAM-1 and E-selectin) in Systemic Sclerosis

This study evaluated whether a new generation antioxidant Activin derived from the grape seed proanthocyanidins, could reduce the induction of the adhesion molecules as a result of inflammatory response in the plasma of systemic sclerosis (SSc) patients. SSc patients were divided into two groups: one group was treated with Activin, a grape seed‐derived proanthocyanidins, while the other group served as control. Patients were given Activin 100 mg/day orally for one month after which the blood samples were withdrawn from both groups of the patients. Blood was also taken from normal human volunteers. Plasma was obtained in fasting state between 8 to 9 A.M. from two groups of SSc patients and controls. Soluble adhesion molecules including ICAM‐1, VCAM‐1, E‐selectin and P‐selectin as well as malonaldehyde, a marker for oxidative stress, were measured. The results of our study demonstrated up‐regulation of these soluble adhesion molecules except for P‐selectin, in the plasma of the SSc patients compared to those obtained from human volunteers. Activin significantly attenuated the increased expression of these adhesion molecules. In addition, there was a significant increase in the amount of malondialdehyde formation in the plasma of the SSc patients, which was also attenuated by Activin. The results of this study demonstrated that Activin could reduce the inflammatory response and the oxidative stress developed in SSc patients.     

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.     

Design, synthesis, and QSAR studies of novel lysine derives as amino-peptidase N/CD13 inhibitors

A series of novel l-lysine derivatives were designed, synthesized, and assayed for their inhibitory activities on amino-peptidase N (APN)/CD13 and matrix metalloproteinase-2 (MMP-2). The preliminary biological test showed that most of the compounds displayed a high inhibitory activity against MMP-2 and a low activity against APN except compound B6 which exhibited good potency (IC(50)=13.2microM) similar with APN inhibitor Bestatin (IC(50)=15.5microM), and could be used as lead compound in the future.