Outcome of hematopoietic stem cell transplant as salvage therapy for Hodgkin's lymphoma in adolescents and young adults at a single institution.

For patients with relapsed Hodgkin's lymphoma (HL), high dose chemotherapy with stem cell rescue (HDCT-SCT) may improve survival over chemotherapy alone. We assessed the outcomes of HDCT-SCT in 37 consecutive adolescent and young adult patients with relapsed HL whose malignancy was categorized based on sensitivity to chemotherapy. We determined whether current outcomes supported the use of HDCT-SCT in all of our patients or just those patients with lower-risk characteristics such as chemosensitivity. With a median follow-up of 6.5 years, the 2-year overall survival (OS) was 89% (95% CI: 62-97%) for the chemosensitive patients (n = 21), whereas for patients with resistant disease (n = 16), OS was 53% (95% CI: 25-74%). Both autologous and allogeneic transplants were well tolerated, with 100-day treatment-related mortality under 10%. Our data show encouraging outcomes for patients with chemosensitive relapsed HL who receive hematopoietic stem cell transplant (HSCT) and support the value of the procedure even when the disease is chemoresistant.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

Antithrombotic efficacy of thrombin inhibitor L-374,087: intravenous activity in a primate model of venous thrombus extension and oral activity in a canine model of primary venous and coronary artery thrombosis.

The small molecule direct thrombin inhibitor L-374,087 was characterized across species in an in vitro activated partial thromboplastin clotting time (aPTT) assay and in vivo in rhesus monkey and dog thrombosis models. In vitro in rhesus, dog, and human plasma, L-374,087 concentrations eliciting 2-fold increases in aPTT were 0.25, 1.9, and 0.28 microM, respectively. In anesthetized rhesus monkeys, 300 microgram/kg bolus plus 12 microgram/kg/min and 300 microgram/kg bolus plus 30 microgram/kg/min L-374,087 i.v. infusions significantly reduced jugular vein thrombus extension, with both regimens limiting venous thrombus extension to 2-fold that of baseline thrombus mass compared with a 5-fold extension observed in the vehicle control group. Antithrombotic efficacy in the rhesus with the lower-dose regimen was achieved with 2.3- to 2.4-fold increases in aPTT and prothrombin time. In a conscious instrumented dog model of electrolytic vessel injury, the oral administration of two 10 mg/kg L-374,087 doses 12 h apart significantly reduced jugular vein thrombus mass, reduced the incidence of and delayed time to occlusive coronary artery thrombosis, and significantly reduced coronary artery thrombus mass and ensuing posterolateral myocardial infarct size. Antithrombotic efficacy in the dog was achieved with 1.6- to 2.0-fold increases in aPTT at 1 to 6 h after oral dosing with L-374,087. These results indicate significant antithrombotic efficacy against both venous and coronary arterial thrombosis with L-374,087 with only moderate elevations in aPTT or prothrombin time. The oral efficacy of L-374,087 characterizes this compound as a prototype for the further development of orally active direct thrombin inhibitors.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin.

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.

Cardiovascular chemotherapy: anticoagulants.

Anticoagulant therapy has changed dramatically during the past two years. Low molecular weight heparin has substantially replaced unfractionated heparin as the parenteral anticoagulant of choice. The use of warfarin has substantially increased, especially for prevention of stroke in the setting of atrial fibrillation. It has become clear that warfarin cannot be administered effectively in an unmonitored fixed-dose fashion. The parenteral direct thrombin inhibitor desirudin was shown to be efficacious in the prevention of deep vein thrombosis in man. Small thrombin and factor Xa inhibitors with in vivo oral anticoagulant activity have been identified.

Identification of low molecular weight GP IIb/IIIa antagonists that bind preferentially to activated platelets.

A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [125I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similar Kd values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (KdActivated = 5 nM and KdResting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selective versus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.

Characterization of the two-step pathway for inhibition of thrombin by alpha-ketoamide transition state analogs.

The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.

Identification and SAR for a selective, nonpeptidyl thrombin inhibitor.

A novel, nonpeptidyl thrombin inhibitor, L-636,619 (1), was identified via topological similarity searching over the Merck Corporate Sample Database. X-ray crystallographic studies determined the geometry for ligand binding to the enzyme. Chemical modification of the P1 and P3 segments of the ligand resulted in enhanced potency and improvement in the chemical stability of the lead. Analog 9 proved to be the most interesting lead from this structurally novel series.

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor.

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.

Kinetic pathway for the slow to fast transition of thrombin. Evidence of linked ligand binding at structurally distinct domains.

The kinetic pathway for the Na+-induced slow --> fast transition of thrombin was characterized. The slow form was shown to consist of two conformers in a 3:1 ratio (ES2:ES1) at 5 degrees C, pH 7.4, Gamma/2 0.3. ES2 binds Na+ 3 orders of magnitude faster than does ES1. The small molecule active site-directed inhibitor L-371,912, and the exosite I binding ligand hirugen, like Na+, bind selectively to ES2 and induce the slow --> fast conversion of thrombin. The slow --> fast transition is limited by the rate of conversion of ES1 to ES2 (k approximately 28 s-1 at 5 degrees C). Replacement of Arg-221a or Lys-224 at the Na+ binding site with Ala appears to selectively alter the slow form and reduce the apparent affinity of the mutants for Na+ and L-371,912. This replacement, however, has little effect on the affinity for the inhibitor in the presence of saturating concentrations of Na+. The kinetically linked ligand binding at the Na+ binding site, exosite I, and the active site of thrombin characterized in the present study indicates the basis for the plasticity of this important enzyme, and suggests the possibility that the substrate specificity and, therefore, the procoagulant and anticoagulant activities of thrombin may be subject to allosteric regulation by as yet unidentified physiologically important effectors.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Alterations in catalytic activity and virus maturation produced by mutation of the conserved histidine residues of herpes simplex virus type 1 protease.

Mutant herpes simplex virus type 1 (HSV-1) viruses were constructed to characterize the roles of the conserved histidine residues (H61 and H148) of HSV-1 protease in the regulation of catalytic activity and virus maturation. Viruses containing mutations at H61 (H61V-V711, H61Y-V715, and H61A-V730) were unable to grow on Vero cells. These mutant viruses could process neither Pra to N0 nor ICP-35cd to ICP-35ef. Transmission electron microscopy studies of H61A-V730-infected Vero cells indicated that capsid maturation is arrested at a state characterized by the predominance of large symmetrical arrays of B capsids within the nucleus. Two mutations at H148 (in viruses H148A-V712 and H148E-V728) gave rise to mutant viruses that grew with a small-plaque phenotype; one of the viruses, H148E-V728, was particularly attenuated when grown at a low multiplicity of infection. The rate of processing of Pra to N0 in infected Vero cells increased in the order H148A-V712 < H148E-V728 < parental strain HSV-1-V731. The observation that H148A-V712 processes Pra to N0 and ICP-35cd to ICP-35ef, whereas H61A does not, establishes H61 as the catalytically essential conserved His assuming that HSV-1 protease, like other serine proteases, utilizes an active-site histidine residue in catalysis. Two of the mutations at H148 (viruses H148K-V729 and H148Y-V716) produced nonviable viruses. H148K-V729 processed neither Pra to N0 nor ICP-35cd to ICP-35ef, whereas H148Y-V716 processed Pra to N0 but did not process ICP-35cd to ICP-35ef. The range of phenotypes observed with the H148 mutant viruses suggests that residue 148 of the HSV-1 protease is a determinant of virus growth rate and viability because of its effects on the activity of the protease and/or the role of the protease domain in capsid assembly and DNA packaging.

A facile system for construction of HSV-1 variants: site directed mutation of the UL26 protease gene in HSV-1.

A 2-plasmid/4-cosmid-based system of mutagenesis is described for construction of herpes simplex virus type 1 (HSV-1) variants with point mutations in the protease gene. The system was used to reconstruct a mutant virus (V701) with Tyr30 to Phe and Ala48 to Val mutations in HSV-1 protease that exhibits the temperature sensitive phenotype of the previously characterized temperature sensitive HSV-1 mutant, ts1201. The 2-plasmid/4-cosmid system of mutagenesis was further validated by using it to construct a virus wherein the active site Ser129 of HSV-1 protease was mutated to Ala. The resulting virus mutant (V713) grew only on the Vero host range cell line PHS-23. In V713 infected Vero cells, the processing of Pra to N(O) was almost completely blocked, and B capsids accumulated in the nucleus in crystal-like aggregates, suggesting that protease activity is required for emergence of monodispersed capsids from these aggregates. Back mutation of Ala129 to Ser using the V713 viral DNA as template for PCR mutagenesis restored the wild-type phenotype verifying that the replicative incompetence of V713 reflected only the effect of the Ser to Ala mutation. The 2-plasmid/4-cosmid system of mutagenesis (and modifications thereof) should facilitate production of new mutant viruses for delineating interactions of domains of HSV-1 protease (as well as other HSV-1 proteins) important for virus replication.

Winter poisoning of coyotes and raptors with Furadan-laced carcass baits.

Three bald eagles (Haliaeetus leucocephalus), a red-tailed hawk (Buteo jamaicensis), and two coyotes (Canis latrans) found in a field in north-central Kansas (USA) in December 1992 were poisoned by flowable carbofuran (Furadan 4F) placed on sheep (Ovis aries) carcasses to kill coyotes. The carbofuran was placed on the carcasses in October 1992, but the coyotes and raptors apparently were killed in late December. Thus, flowable Furadan can cause direct and secondary deaths of wildlife under some circumstances for at least 60 days following placement.

Crystal structure of human alpha-thrombin complexed with hirugen and p-amidinophenylpyruvate at 1.6 A resolution.

Crystals of human alpha-thrombin complexed with hirugen and the alpha-keto acid thrombin inhibitor APPA (p-amidinophenylpyruvate) that diffract to 1.6 A resolution were obtained by soaking an alpha-thrombin-hirugen crystal in a solution of APPA. The crystal structure was determined using the difference Fourier method and refined to an R factor of 18.7% at 1.6 A resolution. This structure is the highest resolution structure of the thrombin molecule that is currently available. With the exception of the region near Arg77A-Asn78, the structures of the thrombin and hirugen molecules in the ternary complex are similar to those reported for the thrombin-hirugen binary complex. As previously determined for the APPA-trypsin complex, the carbonyl carbon atom of APPA forms a covalent bond with O gamma of Ser195 of thrombin to yield a "transition-state" analog of the tetrahedral intermediate. Comparison of the specificity pocket of the APPA complexes of thrombin and trypsin reveals differences in hydrogen bonding and shows for the first time that the S1 site of thrombin is larger than that of trypsin and as a result thrombin may be able to accommodate a bulkier P1 group than trypsin.

Amide and alpha-keto carbonyl inhibitors of thrombin based on arginine and lysine: synthesis, stability and biological characterization.

We report structure-activity investigations in a series of tripeptide amide inhibitors of thrombin, and the development of a series of highly potent active site directed alpha-keto carbonyl inhibitors having the side chain of lysine at P1. Compounds of this class are unstable by virtue of reactivity at the electrophilic carbonyl and racemization at the adjacent carbon (CH). Modifications of prototype alpha-keto-ester 8a have afforded analogs retaining nanomolar Ki. Optimal potency and stability have been realized in alpha-keto-amides 11b (Ki = 2.8 nM) and 11c (Ki = 0.25 nM).