Structure-based virtual screening of small-molecule antagonists of platelet integrin αIIbβ3 that do not prime the receptor to bind ligand

Integrin αIIbβ3 has emerged as an important therapeutic target for thrombotic vascular diseases owing to its pivotal role in mediating platelet aggregation through interaction with adhesive ligands. In the search for effective anti-thrombotic agents that can be administered orally without inducing the high-affinity ligand binding state, we recently discovered via high-throughput screening of 33,264 compounds a novel, αIIbβ3-selective inhibitor (RUC-1) of adenosine-5′-diphosphate (ADP) -induced platelet aggregation that exhibits a different chemical scaffold and mode of binding with respect to classical Arg-Gly-Asp (RGD)-mimicking αIIbβ3 antagonists. Most importantly, RUC-1 and its higher-affinity derivative, RUC-2, do not induce major conformational changes in the protein β3 subunit or prime the receptor to bind ligand. To identify additional αIIbβ3-selective chemotypes that inhibit platelet aggregation through similar mechanisms, we screened in silico over 2.5 million commercially available, ‘lead-like’ small molecules based on complementarity to the predicted binding mode of RUC-2 into the RUC-1-αIIbβ3 crystal structure. This first reported structure-based virtual screening application to the αIIbβ3 integrin led to the identification of 2 αIIbβ3-selective antagonists out of 4 tested, which compares favorably with the 0.003?% “hit rate” of our previous high-throughput chemical screening study. The newly identified compounds, like RUC-1 and RUC-2, showed specificity for αIIbβ3 compared to αVβ3 and did not prime the receptor to bind ligand. They thus may hold promise as αIIbβ3 antagonist therapeutic scaffolds.     

Structural basis for the activation of platelet integrin αIIbβ3 by calcium- and integrin-binding protein 1

Calcium and integrin binding protein 1 (CIB1) is a specific binding partner for the cytoplasmic domain of the αIIb subunit of the highly abundant platelet integrin αIIbβ3. This protein has been suggested to be involved in the regulation of the activation of αIIbβ3, a process leading to platelet aggregation and blood coagulation. In this work, the solution structure of the deuterated Ca(2+)-CIB1 protein complexed with an αIIb peptide was first determined through modern RDC-based NMR methods. Next, we generated a complex structure for CIB1 and the αIIb domain (Ca(2+)-CIB1/αIIb) using the program Haddock, which is based on experimental restraints obtained for the protein-peptide interface from cross-saturation NMR experiments. In this data-driven complex structure, the N-terminal α-helix of the cytoplasmic domain of αIIb is buried in the hydrophobic pocket of the C-lobe of Ca(2+)-CIB1. The C-terminal acidic tail of αIIb remains unstructured and likely interacts with several positively charged residues in the N-lobe of Ca(2+)-CIB1. A potential molecular mechanism for the CIB1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex. Another feature of this work is that, in the NMR cross-saturation experiments, we applied the selective radio frequency irradiation to the smaller binding partner (the αIIb peptide), and successfully detected the binding interface on the larger binding partner Ca(2+)-CIB1 through its selectively protonated methyl groups. This 'reverse' methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope-labeled medium- to large-sized protein are used to study protein-protein interactions.     

In situ construction of ligand nano-network to integrin αvβ3 for angiogenesis inhibition

Angiogenesis occurs during the process of tumor growth, invasion and metastasis, and is essential for the survival of solid tumors. As an integrin significantly overexpressed in human tumor vascular endothelial cells, α_vβ₃ is a suitable targeting site for anti-angiogenesis of tumor. We designed and prepared a self-assembling peptide (SAP) with the ability to targeting α_vβ₃ and self-assembly. SAP formed nanoparticles in solution and transformed into nanofibrous network once specifically binding to integrin α_vβ₃ on the surface of human umbilical vein endothelial cells (HUVECs). The SAP network stably anchored on HUVECs over 24 h, which consequently resulted in high-efficient inhibition of vascularization. In vitro anti-angiogenesis experiment displayed that the inhibition rate of tube-formation reached 94.9%. In vivo anti-angiogenesis array based on chick chorioallantoic membrane (CAM) model exhibited that the SAP had an inhibition rate up to 63.1%. These results indicated the outstanding anti-angiogenic ability of SAP, potentially for tumor therapy.     

Role of α6β4 integrin in cell motility, invasion and metastasis of mammary tumors

Integrin α6β4 is the receptor for the laminin family of extracellular matrix proteins and is widely expressed in most epithelial tissues and Schwann cells. The expression of this integrin is up-regulated in most epithelial tumors, suggesting the role of α6β4 in their progression. The tumor microenvironment is also known to enhance the signaling competence of α6β4 through functional and physical interactions with other receptors. In this review, we discuss the biological mechanisms by which integrin α6β4 promotes carcinoma cell motility and invasion that leads to mammary tumor progression.     

A highly efficient method for the α,β-dehydrogenation of α-amino esters and α-amino-β-diesters

N-monochlorination of N-unprotected α-amino esters and α-amino-β-diesters was efficiently and very simply effected by using inexpensive effervescent sodium dichloroisocyanurate tablets for water disinfection in a biphasic organic solvent-water system. Subsequent dehydrochlorination provided α,β-didehydroaminoacid esters, whose hydrogenation would allow the starting compounds to be easily racemized.     

Analytical techniques for the detection of α-amino-β-methylaminopropionic acid

The non-protein amino acid L-α-amino-β-methylaminopropionic acid (BMAA) has been linked to several neurodegenerative diseases. Its presence in trace amounts in complex sample such as bacterial, plant and mammalian tissue extracts and hydrolyzates makes analysis a complicated process requiring good analytical technique. There are conflicting reports in the literature regarding the presence or absence of BMAA in key samples, but the absence of standardized or validated methods makes comparison of the disparate findings difficult to compare. This critical review will summarize the historic and recent literature, and provide suggestions for improving the methods currently in practice.     

Development of catalysts for the stereoselective hydrogenation of α,β-unsaturated ketones

Iridium phosphinitoxazoline complexes were found to be new efficient catalysts for the asymmetric hydrogenation of arylated α,β-unsaturated ketones. Linear as well as cyclic substrates are hydrogenated with similar success, giving selectivities of up to 99.7% ee.     

A mild, efficient method for the oxidation of α-diazo-β-hydroxyesters to α-diazo-β-ketoesters

A wide variety of α-diazo-β-ketoesters can be prepared in good overall yields via a two-step sequence involving addition of ethyl lithiodiazoacetate to aliphatic, aromatic, and conjugated aldehydes followed by mild oxidation with the Dess-Martin periodinane.     

Evaluation of diverse α/β-backbone patterns for functional α-helix mimicry: analogues of the Bim BH3 domain

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x(L) and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β(3)-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β(3)-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β(3)-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-x(L), affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.     

An efficient dehyrohalogenation method for the synthesis of α,β,β-trifluorostyrenes, α-chloro-β,β-difluorostyrenes and E-1-arylperfluoroalkenes

Dehydrofluorination of 1-aryl-1,2,2,2-tetrafluoroethanes (ArCHFCF₃) and 1-aryl-1-chloro-2,2,2-trifluoroethane (ArCHClCF₃) using lithiumhexamethyldisilazide (LHMDS) in tetrahydrofuran (THF) at room temperature produced 1,2,2-trifluorostyrene and 1-chloro-2,2-difluorostyrene, respectively, in very good isolated yields. Dehydrofluorination of 1,2,2,3,3,3-hexafluoro-1-phenyl-propane (PhCHFCF₂CF₃) and 1,2,2,3,3,4,4,4-octafluoro-1-phenyl-butane (PhCHFCF₂CF₂CF₃) using LHMDS produced the corresponding substituted olefins (1-phenyl-1,2,3,3,3-pentafluoroprop-1-ene and 1-phenyl-1,2,3,3,4,4,4-pentafluorobut-1-ene) in good yield and high E-selectivity. Dehydrofluorination of 1-chloro-1-phenyl-2,2,3,3,3-pentafluoropropane (PhCHClCF₂CF₃) and 1-chloro-1-phenyl-2,2,3,3,4,4,4-heptafluorobutane (PhCHClCF₂CF₂CF₃) produced a mixture of the corresponding E and Z olefins (PhCClCFCF₃ and PhCClCFCF₂CF₃) in good yield.     

Binding of alkaloids into the S1 specificity pocket of α-chymotrypsin: evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K(a) ≈ 10(5) M(-1)) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K(a) ≈ 10(4) M(-1)). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthermore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases.     

Asymmetric photodeconjugation of ammonium ene-carboxylates: temperature effects and evidence for the α-carbon of the dienolic species as a latent trigonal centre

The diastereoselective photodeconjugation of CH₂Cl₂ solutions of enantiopure ammonium ene-carboxylates was studied and the results compared with those of the enantioselective photodeconjugation of ene-esters in the presence of catalytic amounts of chiral aminoalcohols. We have observed a significant entropic effect and shown that a C-β control of the selectivity is operative in the asymmetric protonation of the photodienolic species.     

5-HT1A receptor pharmacophores to screen for off-target activity of α1-adrenoceptor antagonists

The α₁-adrenoceptors (α₁-ARs), in particular the α_1A-AR subtype, are current therapeutic targets of choice for the treatment of urogenital conditions, such as benign prostatic hyperplasia (BPH). Due to the similarity between the transmembrane domains of the α₁-AR subtypes, and the serotonin receptor subtype 1A (5-HT_1A-R), currently used α₁-AR subtype-selective drugs to treat BPH display considerable off-target affinity for the 5-HT_1A-R, leading to side effects. We describe the construction and validation of pharmacophores for 5-HT_1A-R agonists and antagonists. Through the structural diversity of the training sets used in their development, these pharmacophores define the properties of a compound needed to bind to 5-HT_1A receptors. Using these and previously published pharmacophores in virtual screening and profiling, we have identified unique chemical compounds (hits) that fit the requirements to bind to our target, the α_1A-AR, selectively over the off-target, the 5-HT_1A-R. Selected hits have been obtained and their affinities for α_1A-AR, α_1B-AR and 5-HT_1A-R determined in radioligand binding assays, using membrane preparations which contain human receptors expressed individually. Three of the tested hits demonstrate statistically significant selectivity for α_1A-AR over 5-HT_1A-R. All seven tested hits bind to α_1A-AR, with two compounds displaying K _i values below 1 μM, and a further two K _i values of around 10 μM. The insights and knowledge gained through the development of the new 5-HT_1A-R pharmacophores will greatly aid in the design and synthesis of derivatives of our lead compound, and allow the generation of more efficacious and selective ligands.     

1-(1-Alkenyl)benzotriazoles: Novel α-Hydroxyacyl Anion Equivalents for the Synthesis of α-Hydroxy Ketones

α-Lithiated 1-(1-alkenyl)benzotriazoles, generated from the reactions of 1-(1-alkenyl)benzotriazoles with n-BuLi, react with a variety of electrophiles to afford α-substituted 1-(1-alkenyl)benzotriazoles which undergo epoxidation with m-CPBA followed by hydrolysis to give α-hydroxy ketones in good yields. Thus, 1-(1-alkenyl)benzotriazoles behave as α-hydroxyacyl anion equivalents.     

Synthesis of α-substituted iminodiacetate ligands: α-hexadienyl derivatives for the selection of lipoxygenase mimics

Derivatives of iminodiacetic acid (IDA) are important as ligands for metal ions, having numerous applications in separations, sensing, catalysis and medicine. This report describes the preparation of two types of IDA derivatives (1, 2) that could be covalently attached to a polymer or protein surface via a variable length spacer chain. The parent compounds 1 (R′=H) were easily prepared via N-alkylation of dimethyl iminodiacetate with esters of 6-bromo-hexanoic acid and subsequent selective ester hydrolysis. Metal complexes of IDA derivatives having an α-dienyl side chain are required for the selection of histidine-rich proteins with potential lipoxygenase activity. The α-hexadienyl side chain of IDA derivative 2 was selectively introduced in the reaction of (2,4-hexadienyl acetate)Fe(CO)₃ with a glycine-derived TMS-enol ester. Subsequent demetallation, followed by N-carboxymethylation, N-deacylation, N-alkylation with a trichloroethyl 6-halohexanoate, and TCE-ester cleavage provided the desired α-hexadienyl IDA derivative 2. Amide formation with IDA acid 1b demonstrates the feasibility of conjugating the IDA ligands to polymers and proteins while Ni(II)-complexation with the derived IDA triacid 1e shows the complexing ability of the tethered IDA ligand.     

Determination of the absolute configuration of two αvβ6 integrin inhibitors for the treatment of idiopathic pulmonary fibrosis and investigations on the asymmetric 1,4-addition of arylboronic acids to crotonate esters bearing a C4-oxygen substituent

The absolute configuration of two novel α_vβ₆ integrin inhibitors was established via degradation to the corresponding C3-aryl substituted butyrolactone. The configuration of the resulting lactones was established by asymmetric synthesis using 1,4-addition of arylboronic acids to butenolide, catalysed by bis(norbornadiene)rhodium (I) tetrafluoroborate in the presence of (R)-BINAP, and confirmed by X-ray crystallography. Studies on arylboronic acid conjugate additions to acyclic crotonate esters bearing a γ-oxygen substituent are also reported. Three Rh catalysts were investigated and the one giving the highest enantioselectivity was bis(norbornadiene)rhodium (I) tetrafluoroborate.     

Thermodynamic Data for the Formation of 1:1 and 2:1 Complexes of α,ω-Diamino Dihydrochlorides with 18-Crown-6 in Aqueous Solution

Calorimetric titrations are used to study the interactions between the crown ether 18-crown-6 and several α,ω-diamino dihydrochlorides in aqueous solution. These complexes are formed by ion-dipole interactions between the positively charged nitrogen atoms and the oxygen donor atoms of the crown ether. Depending on the experimental conditions, the formation of 1:1 or 2:1 complexes (ligand:diamines) can be studied. The solvation of the ligand and the amines are responsible for the observed thermodynamic values. The number of water molecules released during the reaction were calculated from the determined reaction entropies. Formation of 1:1 complexes distorts the solvation shell around the molecules. As a result, the number of solvent molecules released during the formation of the 2:1 complexes is slightly smaller than the number released from formation of the 1:1 complex. No experimental evidence is observed for the formation of complexes between one crown ether and two protonated amino groups.     

β-Diketiminate complexes of Group 4: active complexes for the isomerization of α-olefins and the polymerization of propylene towards elastomeric polypropylene

The β-diketiminate lithium ligand [{N(SiMe₃)C(Ph)}₂CH][Li] reacted with Group 4 metal salts (Ti and Zr) to yield the complexes [{N(SiMe₃)C(Ph)}₂CH]₂TiCl₂ (5) and [{N(SiMe₃)C(Ph)}₂CH][N(SiMe₃)C(Ph)NC(Ph)CH(SiMe₃)]ZrCl₂ (6). The crystal structure of 6 shows that one of the two ketamidinate ligands undergoes an isomerization to the corresponding substituted benzamidinate. A mechanism for the catalyzed isomerization of the β-diketiminate ligand is presented. Complex 5 was found to be active in the polymerization of propylene, producing remarkably high-molecular weight (>100,000 g mol⁻¹) elastomeric polymer, whereas the zirconium complex was found inactive. Complex 5, and surprisingly complex 6, were found to be active catalysts, in the presence of MAO (methylalumoxane), for the isomerization of aliphatic olefins (1-octene, allylbenzene). Each complex catalyzed the olefins by different mechanisms. Kinetic studies for the isomerization of allylbenzene by complex 5 show that the reaction rate follows a first order in both catalyst and olefin concentrations with ΔH^‡=14.7(4) kcal mol⁻¹ and ΔS^‡=−33(1) e.u. These findings support the epimerization mechanism of the last inserted monomer that is proposed for as the primary cause for the elastomeric properties of polypropylene produced by this complex. In addition, we show how complementary isomerization studies of α-olefins shed light on the polymerization mechanism.