Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH₂. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH₂, competitively inhibits α-thrombin''s cleavage of the S2238 chromogenic substrate with a K_i of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.     

Rational design of potent human transthyretin amyloid disease inhibitors

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein-drug complexes have been determined to allow detailed analyses of the protein-drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4, 6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.     

Rational design of a thrombin electrochemical aptasensor by conjugating two DNA aptamers with G-quadruplex halves

A novel strategy for the fabrication of an electrochemical aptasensor is proposed; this strategy has been employed in this work to assay thrombin concentration. Two well-designed oligonucleotides were used as the core element. G-quadruplex–hemin complexes can be formed on the surface of the electrode to give a detectable signal only when thrombin is not bound to the aptamers. The detection limit of the biosensor has been lowered to 10 nM. Moreover, since the electroactive probe is not required to be bound to the oligonucleotide, this strategy may integrate the advantages of being both label-free and cost-effective.     

The design of potent and selective inhibitors of thrombin utilizing a piperazinedione template: part 1.

Utilizing X-ray crystallography and molecular modeling, highly potent and selective peptidomimetic thrombin inhibitors have been designed containing a rigid piperazinedione template. The synthesis and biological activity of these compounds will be described.     

The design of potent and selective inhibitors of thrombin utilizing a piperazinedione template: part 2.

Potent and selective thrombin inhibitors have been prepared with a piperazinedione template and L-amino acids. Likewise, incorporation of D-amino acids led to potent inhibitors with a novel mode of binding. Herein, the structure activity relationships and structural aspects of these compounds will be described.     

Rational design and synthesis of potent aminoglycoside antibiotics against resistant bacterial strains

Based on the structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construction of kanamycin derivatives, in which the C4′-position on ring I of neamine moiety was modified, was developed. Most synthetic analogues exhibited good to excellent antibiotic activity against some typical drug-resistant bacteria. The disclosed results suggested that the C4′-position of aminoglycosides such as kanamycin may be an ideal site for modification to gain new modifying enzyme-resistant aminoglycoside antibiotics.     

Rational design of novel, potent piperazinone and imidazolidinone BACE1 inhibitors

Guided by structure-based design, we synthesized two novel series of potent inhibitors of BACE1 and generated extensive SAR around both the prime and non-prime side binding pockets. The key feature of both series is a cyclic amine motif specifically crafted to achieve interactions with both the flap and with the S2' pocket.     

Rational design and synthesis of highly potent anti-acetylcholinesterase activity huperzine A derivatives

By targeting multi-active sites of acetylcholinesterase (AChE), a series of huperzine A (Hup A) derivatives with various aromatic ring groups were designed and synthesized by Schiff reaction. They were evaluated as AChE and butyrylcholinesterase (BChE) inhibitors. Results showed very significant specificity that the group of imine derivatives could inhibit TcAChE and hAChE, but no inhibitory effect on hBChE was detected. The experiment was explained by a docking study. In the docking model, we confirmed that aromatic ring of Hup A derivatives played the π–π stacking against aminophenol residues of AChE, and the structure–activity relationship (SAR) was discussed.     

Rational design of transition-state analogues as potent enzyme inhibitors with therapeutic applications.

The structures of the transition states for a variety of enzyme-catalyzed ribosyl group transfer reactions, determined by computational evaluation of multiple tritium and heavy atom kinetic isotope effects on these enzymatic reactions, have been found to show a considerable variation in the extent of bond cleavage at the ribosyl anomeric carbon. The calculated transition-state structures have been used to guide the design of high-affinity transition-state analogue inhibitors for 5'-methylthioadenosine nucleosidases with potential as therapeutic agents.     

Rational design of potent and selective NH-linked aryl/heteroaryl cathepsin K inhibitors

Prior reports from our laboratories have identified the nonpeptidic inhibitor 2 as a potent and selective Cathepsin K (Cat K) inhibitor. Modelling studies suggested that the introduction of a NH linker between the P3 aryl and P2 leucinamide moieties would allow the formation of a H-bond with the Gly66 residue of Cat K, hopefully increasing potency. Aniline 4 was thus synthesized and showed improved potency over its predecessor 2. Further modelling concluded that a 2-substituted five membered ring could more adequately place the P3 moiety of 4 into the S3 pocket of Cat K. The synthesis of the 2-substituted thiophene 5 confirmed this hypothesis by displaying a slight increase in potency against Cat K (>10-fold increase in potency vs 2) and a good selectivity profile against Cathepsins B, L, and S. This rationally designed inhibitor 5 also displayed increased potency in a functional bone resorption assay (10nM) versus 2 (95 nM).     

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.     

Crystal structure of the anticoagulant slow form of thrombin

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.     

The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo

Administration of the thrombin mutant W215A/E217A (WE), rationally designed for selective activation of the anticoagulant protein C, elicits safe and potent anticoagulant and antithrombotic effects in a baboon model of platelet-dependent thrombosis. The lowest dose of WE tested (0.011 mg/kg bolus) reduced platelet thrombus accumulation by 80% and was at least as effective as the direct administration of 40-fold more (0.45 mg/kg bolus) activated protein C. WE-treated animals showed no detectable hemorrhage or organ failure. No procoagulant activity could be detected for up to 1 week in baboon plasma obtained following WE administration. These results show that engineered thrombin derivatives that selectively activate protein C may represent useful therapeutic agents for the treatment of thrombotic disorders.     

Rational design and synthesis of potent and long-lasting glutamic acid-based dipeptidyl peptidase IV inhibitors

A series of (2S)-cyanopyrrolidines with glutamic acid derivatives at the P2 site have been prepared and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV). The structure–activity relationships (SAR) led to the discovery of potent 3-substituted glutamic acid analogues, providing enhanced chemical stability and excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. Compound 13f exhibited the ability to both significantly decrease the glucose excursion and inhibit plasma DPP-IV activity.     

A short synthesis of argatroban. a potent selective thrombin inhibitor.

Argatroban was synthesized in seven steps from 4-methylpiperidine. The condensation of (+/-)-trans-benzyl 4-methylpipecolic acid ester with N(alpha)-Boc-N(omega)-nitro-L-arginine led to two diastereomers that were separated. One of them is the precursor of argatroban.     

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues.

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17, Lys21]hCt (1), which had a 5-10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We hypothesized that a stabilized, possibly type I beta turn/beta sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD spectra indicated increased type I beta turn and antiparallel beta sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in 'freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a beta turn/beta sheet conformation in region 17-21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt-based drug for the treatment of osteoporosis and other bone-disorder-related diseases.     

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element.

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.     

SSR182289A, a selective and potent orally active thrombin inhibitor

SSR182289A 1 is the result of a rational optimisation process leading to an orally active thrombin inhibitor. The structure incorporates an original 2-(acetylamino)-[1,1'-biphenyl]-3-sulfonyl N-terminal motif, a central l-Arg surrogate carrying a weakly basic 3-amino-pyridine, and an unusual 4-difluoropiperidine at the C-terminus. Its synthesis is convergent and palladium catalysis has been employed for the construction of the key C-C bonds: Suzuki coupling for the bis-aryl fragment and Sonogashira reaction for the delta- bond of the central amino-acid chain. The compound is a potent inhibitor of thrombin's activities in vitro and demonstrates potent oral anti-thrombotic potencies in three rat models of thrombosis. The observed in vitro potency could be rationalized through the examination of the interactions within the SSR182289A 1 - thrombin crystal structure. SSR182289A 1, has been therefore selected for further development.