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Biomedical subjects

R S Alexander

Publications and source records attributed to R S Alexander.

At least 19 recordsLinked to original sources
PubMed2001

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

Administration, Oral↗
PubMed2000

Design, synthesis, and biological evaluation of potent and selective amidino bicyclic factor Xa inhibitors.

Thrombotic diseases are a major cause of death and morbidity. Factor Xa (fXa) plays a vital role in the regulation of normal homeostasis and abnormal intravascular thrombus development in the blood coagulation cascade. A novel series of fXa inhibitors incorporating an amidino 6,5-fused bicyclic moiety at the P1 position has been designed and synthesized based on molecular modeling studies. Structure-activity relationship (SAR) studies have led to selective subnanomolar fXa inhibitors. The most potent fXa inhibitor in this series (72, SE170) has a potent inhibition constant (K(i) = 0.3 nM), is 350-fold selective for fXa over trypsin, and also shows good in vivo efficacy in a rabbit arterio-venous thrombosis model (ID(50) = 0.14 micromol/kg/h). An X-ray crystal structure of 72 complexed to bovine trypsin was completed, and a binding mode of 72 with fXa has been proposed based on modeling with human des-Gla-fXa.

Amidines↗
PubMed1999

Design and synthesis of isoxazoline derivatives as factor Xa inhibitors. 2.

Intravascular clot formation is an important factor in a number of cardiovascular diseases. Therefore, the prevention of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (FXa), the enzyme directly responsible for thrombin activation. Herein we report a series of isoxazoline derivatives which are potent FXa inhibitors. Optimization of the side chain at the quaternary position of the isoxazoline ring led to SK549 which showed subnanomolar FXa potency (K(i) 0.52 nM). SK549 shows good selectivity for FXa compared to thrombin and trypsin, potent antithrombotic effect in the rabbit arterio-venous thrombosis model, and improved pharmacokinetics relative to other compounds evaluated from this series.

Animals↗
PubMed1999

Design and synthesis of potent and selective 5,6-fused heterocyclic thrombin inhibitors.

Thrombin, a serine protease, plays a central role in the initiation of thrombotic events. We report the design, synthesis, and antithrombotic efficacy of XU817 (7), a nonpeptide 5-(amidino) indole thrombin inhibitor. Utilizing the co-crystal structure of XU817 bound in the active site of thrombin we were able to synthesize analogs with enhanced thrombin affinity.

Amidines↗
PubMed1999

Crystallization and preliminary X-ray diffraction data of the complex of recombinant tick anticoagulant peptide (rTAP) and bovine factor Xa.

The complex of bovine factor Xa and recombinant tick anticoagulant peptide (rTAP) was crystallized in two different crystal forms using polyethylene glycol as a precipitant. Form I belongs to space group P42212 with unit-cell dimensions a = b = 133.1, c = 68.8 A. It contains one complex per asymmetric unit and diffracts to 3.0 A resolution. Form II belongs to P41212 (or P43212) with dimensions a = b = 126.5, c = 146.7 A; it contains two complexes per asymmetric unit and diffracts to 2.5 A. The crystals of both forms consist of factor Xa (MW = 45.3 kDa) and rTAP (MW = 6.7 kDa).

Animals↗
PubMed1998

Rational design of boropeptide thrombin inhibitors: beta, beta-dialkyl-phenethylglycine P2 analogs of DuP 714 with greater selectivity over complement factor I and an improved safety profile.

The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that appear to be related to its ability to inhibit complement factor I, thereby activating the complement cascade. Using X-ray crystal structure information, we have designed compounds that have greater selectivity for thrombin over factor I and that have reduced tendency to produce these side effects.

Animals↗
PubMed1998

Unexpected binding mode of tick anticoagulant peptide complexed to bovine factor Xa.

The structure of recombinant tick anticoagulant peptide (rTAP) complexed to bovine factor Xa at 3.0 A resolution reveals the structural basis for the specificity and the high affinity of rTAP. Three N-terminal residues, Tyr501, Asn502 and Arg503, play a critical role in the complex formation as suggested by earlier mutagenic studies and the ornithodorin-thrombin complex. Unexpectedly, the side-chain of Tyr501 is located in the S1 pocket, although factor Xa favors arginine as a P1 residue. Arg503 is located at the aryl binding pocket and forms a salt-bridge with Glu97 of factor Xa. The autolysis loop, which is disordered in the uninhibited factor Xa structure, is involved in the formation of the complex as a part of the secondary binding site. The C-terminal helix of rTAP interacts with factor Xa as a secondary binding determinant. The N-terminal residues of rTAP reorganize during the formation of the factor Xa-rTAP complex from the conformation found in the solution into an extended conformation. The presence of the secondary binding site confirms the proposed two-step kinetic mechanism based on the results of a mutagenesis study.

Animals↗
PubMed1997

New inhibitors of thrombin and other trypsin-like proteases: hydrogen bonding of an aromatic cyano group with a backbone amide of the P1 binding site replaces binding of a basic side chain.

Highly effective thrombin inhibitors have been obtained by preparing boronic acid analogues of m-cyano-substituted phenylalanine and its incorporation into peptides. The cyano group enhances binding by several orders of magnitude. For example, Ac-(D)Phe-Pro-boroPheOH binds to thrombin with a Ki of 320 nM and the Ki of Ac-(D)Phe-Pro-boroPhe(m-CN)-OH is 0.79 nM. Protein crystal structure determination of trypsin complexed to H-(D)Phe-Pro-boroPhe(m-CN)-OH indicates that the aromatic side chain is bound in the P1 binding site and that the cyano group can act as a H-bond acceptor for the amide proton of Gly219. Enhanced binding for inhibitors containing the m-cyano group was observed for coagulation factor Xa and for the factor VIIa.tissue factor complex [Ki values of Ac-(D)Phe-Pro-boroPhe(mCN)-OH are 760 and 3.3 nM, respectively]. This result is consistent with the sequence homology of these two enzymes in the P1 binding site. Two enzymes lacking the strict homology in the P1 binding site, pancreatic kallikrein and chymotrypsin, did not exhibit significantly enhanced binding.

Amides↗
PubMed1995

Structure of His94-->Asp carbonic anhydrase II in a new crystalline form reveals a partially occupied zinc binding site.

The structure of histidine 94-->aspartate (H94D) carbonic anhydrase II (CAII) crystallized in an orthorhombic space group has been determined to 2.5 A resolution. This crystal form is not isomorphous with monoclinic wild-type enzyme crystals or with the monoclinic crystal form of H94D CAII reported earlier [Kiefer,L.L., Ippolito, J.A., Fierke, C.A. and Christianson, D.W. (1993) J. Am. Chem. Soc., 115, 12581-12582]. In monoclinic H94D CAII, a fully occupied zinc ion is tetrahedrally coordinated by D94, H96, H119 and a water molecule. In orthorhombic H94D CAII, a partially occupied zinc ion is coordinated by H96 and H119 and only weakly coordinated by a disordered D94 side chain. These differences are particularly surprising given that the two crystal forms co-precipitate in the same drop in the same experiment. Re-refinement of the orthorhombic crystal form of H94C CAII and comparison with its corresponding monoclinic crystal form yield similar results. It appears that partial-but not full-zinc dissociation accompanies the crystallization of CAII variants in the orthorhombic crystal form, and significant differences on the protein surface presumably affect the relative stability of each crystal lattice. These results underscore an unexpected ambiguity in this protein engineering experiment: which crystal structure of H94D CAII should be correlated with functional measurements made in solution?

Binding Sites↗
PubMed1994

Identification of two hydrophobic patches in the active-site cavity of human carbonic anhydrase II by solution-phase and solid-state studies and their use in the development of tight-binding inhibitors.

This paper describes inhibitors for human carbonic anhydrase II (HCAII, EC 4.2.1.1) that bind with nanomolar dissociation constants. These inhibitors were developed by exploiting interactions with hydrophobic "patches" in the lip of the active site of this enzyme. These patches are molecular surfaces presented by a phenylalanine on one face of the active-site cleft (Phe-131) and three adjacent hydrophobic residues on the opposite face (Leu-198 and Pro-201/202). Comparison of the affinities of molecules that can occupy either one or both of the two sites indicates that these hydrophobic interactions can contribute factors of 10(2)-10(3) to binding constants and that the strength of the interaction is relatively insensitive to the structure of the hydrophobic ligand. One of these inhibitors, the competitive inhibitor N-[N-[N-(4-sulfamoylbenzoyl)phenylglycyl]glycyl]glycine benzyl ester (17), has been studied by X-ray crystallographic methods in its complex with HCAII at 1.9-A resolution. The geometry of binding of the arylsulfonamide group of 17 is similar to geometries observed in other HCAII-arylsulfonamide complexes. The aromatic side chain of the phenylglycine residue of the inhibitor is inferred to pack against the hydrophobic Phe-131 face, and this interaction "steers" the peptide backbone of the inhibitor toward a region in the HCAII active site different from that occupied in the related triglycyl peptide. Attempts to design inhibitors capable of binding simultaneously to Phe-131 and Leu-198/Pro-201/202 did not lead to molecules that bound more tightly than those binding to these hydrophobic sites individually.

Amino Acid Sequence↗
PubMed1994

Positions of His-64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors.

The 3-dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, 1a, 1b, and 1c, has been determined by X-ray crystallographic methods. The 3 inhibitors (1a = C8H12N2O4S3) vary only in the length of the substituent on the 4-amino group: 1a, proton; 1b, methyl; and 1c, ethyl. The binding constants (Ki's) for 1a, 1b, and 1c to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors 1a and 1b, electron density can be observed for His-64 and a bound water molecule in the native positions. When inhibitor 1c is bound, the side chain attached to the 4-amino group is positioned so that His-64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason 1c binds tighter to HCAII than does 1a or 1b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His-64 side chain in a higher energy state.

Carbonic Anhydrase Inhibitors↗
PubMed1993

Engineering the zinc binding site of human carbonic anhydrase II: structure of the His-94-->Cys apoenzyme in a new crystalline form.

The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155. This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions). Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A. Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94. Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy. However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation. Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Structural comparison of sulfodiimine and sulfonamide inhibitors in their complexes with zinc enzymes.

The three-dimensional structure of (L(-)-2-carboxy-3-phenylpropyl) methylsulfodiimine in its complex with the zinc metalloenzyme carboxypeptidase A has been determined at 2.25-A resolution by x-ray crystallographic methods. This is the first example of a sulfodiimine-containing inhibitor binding to a zinc enzyme, and the structure of the enzyme-inhibitor complex reveals that the tetrahedral sulfodiimine group coordinates to the active site zinc ion in unidentate fashion. The zinc-coordinated nitrogen atom of the sulfodiimine group is also within hydrogen bonding distance to active site base Glu-270; presumably, the sulfodiimine is ionized and accepts a hydrogen bond from protonated Glu-270. The other sulfodiimine nitrogen accepts a hydrogen bond from Arg-127, and the inhibitor binds as a possible analogue of the tetrahedral transition state (or intermediate) in a promoted water pathway for peptide hydrolysis. The unidentate sulfodiimine-zinc binding mode observed in this enzyme-inhibitor complex is reminiscent of that observed in sulfonamide complexes with the zinc metalloenzyme carbonic anhydrase II, and the structural features of sulfodiimine- and sulfonamide-zinc interactions exhibit important similarities among recently determined structures of enzyme-inhibitor complexes: ionized nitrogens bind to zinc in each structure, and these nitrogens are engaged in hydrogen bond interactions with neighboring enzyme residues.

Binding Sites↗