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Purpose: Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors., Experimental Design: We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells., Results: Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo., Conclusions: Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

Objective: Based on promising in vitro and in vivo activity of several histone deacetylase inhibitors in Hodgkin lymphoma (HL), we investigated SNDX-275, an oral class 1 isoform-selective histone deacetylase inhibitors in HL-derived cell lines., Materials and Methods: Proliferation and cell death were examined by MTS assay, Annexin V/propidium iodide, and fluorescence-activated cell sorting analysis. Gene and protein expression were measured by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical analysis. A multiplex assay was used to determine cytokines and chemokines., Results: SNDX-275 induced cell death in a dose- and time-dependent manner with an IC(50) at the sub- and lower micromolar range at 72 hours. At the molecular level, SNDX-275 increased histone H3 acetylation, upregulated p21 expression, and activated the intrinsic apoptosis pathway by downregulating the X-linked inhibitor of apoptosis protein. SNDX-275 downregulated expression of antiapoptotic Bcl-2 and Bcl-xL proteins without altering Mcl-1 or Bax levels. Combination studies demonstrated that two Bcl-2 inhibitors (ABT-737 and obatoclax) significantly enhanced the effect of SNDX-275. SNDX-275 modulated the level of several cytokines and chemokines, including interleukin-12 p40-70, interferon-inducible protein-10, RANTES (regulated on activation, normal T expressed and secreted), interleukin-13, interleukin-4, and thymus and activation-regulated chemokine and variably induced the cancer/testis antigen expression of MAGE-A4 and survivin in HL cell lines., Conclusions: SNDX-275 has antiproliferative activity in HL cell lines, involving several mechanisms: induction of apoptosis, regulation of cytokines and chemokines, and alteration of cancer/testis antigens. Clinical investigation of SNDX-275 alone or in combination with Bcl-2 inhibitors is warranted in patients with HL. Phase 2 studies with SNDX-275 in HL are ongoing, and future clinical studies should investigate combinations with SNDX-275., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Investigations of regional genetic differentiation are essential for describing phylogeographic patterns and informing management efforts for species of conservation concern. In this context, we investigated genetic diversity and evolutionary relationships among great gray owl (Strix nebulosa) populations in western North America, which includes an allopatric range in the southern Sierra Nevada in California. Based on a total dataset consisting of 30 nuclear microsatellite DNA loci and 1938-base pairs of mitochondrial DNA, we found that Pacific Northwest sampling groups were recovered by frequency and Bayesian analyses of microsatellite data and each population sampled, except for western Canada, showed evidence of recent population bottlenecks and low effective sizes. Bayesian and maximum likelihood phylogenetic analyses of sequence data indicated that the allopatric Sierra Nevada population is also a distinct lineage with respect to the larger species range in North America; we suggest a subspecies designation for this lineage should be considered (Strix nebulosa yosemitensis). Our study underscores the importance of phylogeographic studies for identifying lineages of conservation concern, as well as the important role of Pleistocene glaciation events in driving genetic differentiation of avian fauna., (Published by Elsevier Inc.)

Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor-associated antigens (TAAs), which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. Importantly, while these cells showed increased resistance to gemcitabine, a commonly used drug for the treatment of refractory HL, HL SP cells also expressed higher levels of the TAAs MAGEA4, SSX2, survivin, and NY-ESO-1, which allowed them to be specifically recognized and killed by TAA-specific cytotoxic T lymphocytes. This study suggests that chemoresistant HL SP cells can be targeted by the immune system, providing a rationale for combined chemotherapy and immunotherapy for the treatment of HL.

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.

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

Background: Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation., Methods and Results: The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor-derived peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of approximately 64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT., Conclusions: These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.

Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa (fXa), a central enzyme in the blood clotting cascade. As such, TAP is a potential antithrombotic agent. Site-directed mutagenesis studies were undertaken to determine the feasibility of increasing the inhibitory potency of TAP toward fXa. The amino acid substitutions Tyr-1 to Trp (Y1W) and Asp-10 to Arg (D10R) increased inhibitory potency toward human fXa by 2.5- and 4-fold, respectively. The increased inhibitory potency reflected a decrease in the rate constant for dissociation of the final fXa-TAP inhibitory complex. The double mutant, Y1W/D10R, exhibited an inhibition constant of 10 pM, a 37-fold enhancement of inhibitory potency toward human fXa. The improvement in inhibitory potency was less pronounced (12-fold) with dog fXa wherein Kis of 220 and 18 pM were observed for wild-type TAP and the double mutant, respectively. Mutation of Tyr-1 to Glu (Y1E) generated a weaker inhibitor (Ki = 2 nM) that bound human fXa more slowly. However, no change in inhibitory potency toward human fXa was observed when Tyr-1 was replaced by Phe. Taken together, these observations are consistent with the view that a hydrophobic amino acid at the N-terminus of TAP may be a determinant of inhibitory potency. Decreases by 3-4 orders of magnitude in inhibitory potency were noted upon mutation of Asn-2 and Leu-4 of TAP, further implicating the N-terminal domain as an important determinant of inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.

Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km values of the purified native enzyme are 2.0 min-1 and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.

The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.

HIV-1 reverse transcriptase can catalyze the addition of either azidothymidine monophosphate (AZTMP) or thymidine monophosphate (dTMP) to a primer strand opposite template adenosine bases. The ratio of incorporation of AZTMP to dTMP as catalyzed by HIV-1 reverse transcriptase has been determined to be 0.4 using an RNA-DNA duplex substrate prepared from oligonucleotides with sequences taken from the HIV-1 genome sequence. Slight variations are found for the incorporation ratio of the two nucleotides on other substrates. Substrates containing more than one adenosine in the single-stranded part of the template allow for more chances to incorporate AZTMP and less full-length product. Variations in the intensity of bands on an autoradiograph of a DNA sequencing gel corresponding to different positions of incorporation of AZTMP suggest that not all template adenosine positions offer the same level of discrimination against incorporation of AZTMP. A reverse transcriptase containing a set of four mutations (D67N, K70R, T215Y, K219Q) known to cause resistance to AZT in cell culture assays has a ratio of incorporation that is 0.77 +/- 0.03 times the ratio for the wild-type reverse transcriptase opposite one specific template adenosine. In contrast, a hybrid mutant containing the same four mutations that cause resistance to AZT and an additional mutation, Y181C, which by itself causes resistance to the non-nucleoside inhibitor L-697,661 [Sardana et al. (1992), J. Biol. Chem. 267, 17526-17530], has a ratio of incorporation that is 1.34 +/- 0.01 times that of the wild-type, indicating that the hybrid mutant enzyme is more susceptible to inhibition by AZTTP than the wild-type reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)

The therapeutically important anticoagulant heparin catalyzes inactivation of thrombin by antithrombin III via formation of an intermediary ternary thrombin-heparin-antithrombin III complex that is subsequently converted to a stable thrombin-antithrombin III complex with the release of heparin. Point mutations at Arg-180, Arg-245, Lys-248, and Lys-252 in thrombin markedly reduced the efficiency of heparin catalysis by decreasing the stability of the ternary intermediate, whereas the inactivation of thrombin by antithrombin alone was not affected by these mutations. These results together with an analysis of the x-ray crystal structure of thrombin yielded a model for the thrombin-heparin interaction, wherein heparin forms salt linkages along a groove in thrombin defined by Lys-252, Lys-248, Arg-245, Arg-89, Arg-98, and Arg-180.

The kinetics and equilibrium properties were investigated for the interconversion between the active dimer of human immunodeficiency virus 1 (HIV-1) protease and its inactive monomeric subunits. The equilibrium dissociation constant (Kd) of the dimeric protease as well as the monomer association rate were obtained by monitoring the fluorescence change of an active-site-directed fluorescent probe (L-737244) upon its binding to the protease. The Kd of the HIV-1 protease is strongly pH dependent. At pH 5.5 where the enzyme is most active catalytically, the extrapolated values of Kd are 0.75 and 3.4 nM at 30 and 37 degrees C, respectively. The rate constant for HIV-1 monomer association, approximately 4 x 10(5) M-1 s-1, is within the range commonly observed for protein-protein interactions. Dimer dissociation was further scrutinized in the presence of an inactive, point mutant form of the enzyme. As a result of subunit exchange between the native and mutant enzymes and the formation of an inactive heterodimer, there was a time-dependent decrease in the activity of the native protease. Enzyme activity could be reinstated with the addition of an active-site-directed inhibitor (L-365862) which selectively binds active dimers. The rate of dimer dissociation was found to also decrease with pH. At pH 5.5 and 30 degrees C, the half-life for subunit dissociation is about 0.5 h. The slow dissociation, coupled with the high stability for dimer association, attests to the importance of allowing sufficient time for dimer-monomer equilibration in kinetic assays in order to avoid reaching erroneous conclusions in studies of dimer dissociation.

rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts. Oligomerization of rev is thought to be associated with RNA binding and rev function. Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation. rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations. The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal [K = 1.08 x 10(6) M-1 at 4 degrees C]. The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9. Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy. Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay. rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function.

A high level of plasma fibrinogen has been shown to be an important risk factor for myocardial infarction and stroke. Thus, we were prompted to investigate regulation of human fibrinogen biosynthesis, a process wherein expression of the B beta-chain of fibrinogen appears to be rate-limiting for fibrinogen secretion. Using electrophoretic mobility shift assays with synthetic probes representing portions of the human B beta-fibrinogen promoter, we have defined several elements that bind distinct classes of transcription factors present in human hepatoma cell nuclear extracts. The contribution of each element to promoter activity was demonstrated in transfection experiments using promoter-chloramphenicol acetyltransferase constructs and human hepatoma cells. Our observations indicate that two distinct sequence elements are required for maximal induction of transcription by interleukin-6. One of these sequences is an IL-6-RE core element similar to that reported for the rat alpha 2-macroglobulin promoter and the other is a binding site for the C/EBP family of transcription factors. We also report two additional elements, one negative- and one positive-acting, that bind novel sequence-specific factors.

The catalytic utilization of dimethylallyl, geranyl, farnesyl, and geranylgeranyl diphosphates in the reaction catalyzed by recombinant human farnesyl:protein transferase (hFPTase) has been examined in the presence of three different protein substrates, Ras-CVLS, Ras-CVIM, and Ras-CAIL. hFPTase catalyzed both farnesylation and geranylation of Ras-CVLS and of Ras-CVIM but not of Ras-CAIL. Geranylgeranylation was observed, but only when Ras-CVIM was the acceptor substrate. Steady-state initial velocity and dead-end inhibitor studies indicate that hFPTase-catalyzed geranylation, like bovine FPTase-catalyzed farnesylation, proceeds through a random order, sequential mechanism. Surprisingly, however, Michaelis constants for a given protein acceptor substrate varied depending upon which isoprenoid diphosphate was used as the donor substrate, showing that these substrates do not bind independently to the enzyme (under catalytic conditions). In addition, at very high concentrations of Ras-CVIM, substrate inhibition was observed in the presence of both FPP and GPP. Isotope partitioning studies showed that, at high concentrations of Ras-CVIM, more than 80% of the bound farnesyl diphosphate (FPP) can be trapped as product, suggesting that the binary complex is catalytically competent and that the ternary complex proceeds to product faster than it releases FPP. The release rate of FPP from the binary complex was calculated to be 0.05 s-1, which is only about eight times greater than kcat. Thus, the binding of FPP to the enzyme in the presence of the protein substrate is not an equilibrium situation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.

To demonstrate that human alpha-thrombin is effectively inactivated by human antithrombin III (AT) during the production of a fibrin clot we measured the amount of alpha-thrombin activity which can be recovered from a clot generated from purified human proteins. We discovered that 0.05-0.07% of the original alpha-thrombin activity is recovered from a fibrin clot produced from a reaction mixture where the initial concentrations of AT and alpha-thrombin were chosen at a ratio (17.5) to allow complete conversion of fibrinogen to fibrin. These results indicated that alpha-thrombin is successfully inactivated by AT during the production of a fibrin clot. Further, when an amount of alpha-thrombin equal to that recovered from a fibrin clot is introduced into a solution of fibrinogen and AT identical to that utilized to produce the clot only 4% of the fibrinogen is converted to fibrin. These results suggest that i) when a fibrin clot is dissolved during fibrinolytic therapy little active alpha-thrombin should be released from the clot and ii) this amount of thrombin is insufficient to catalyze rethrombosis without proposing de novo production of thrombin. The action on factors XI, VIII, and V of the small amount of thrombin released upon thrombolysis, however, may provide the stimulus for de novo production of sufficient thrombin to catalyze rethrombosis.

The inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by pyridinone compounds has been investigated using as templates synthetic RNA with sequences based on the HIV-1 genome sequence. In reactions catalyzed by the enzyme that incorporated more than one nucleotide per primer, inhibition by a representative pyridinone inhibitor, 3-[2-(1,3-benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2(1H)one (L-696,229), was noncompetitive against deoxynucleotide triphosphate. For reactions that incorporated one deoxynucleotide per primer, IC50 values ranged from 20 to 200 nM, depending on the position of incorporation of the incoming deoxynucleotide base on the template. Inhibition of synthesis on a set of four templates differing only at the template base complementary to the incoming nucleotide had similar IC50 values. These results demonstrate that inhibitory potency is dependent on the primary structure of the template and that inhibitory potency is largely independent of the identity of the incoming nucleotide base. The inhibition of HIV-1 reverse transcription by L-696,229 also displayed slow-binding characteristics. The slow-binding aspect was exploited to gauge the interaction between inhibitor and enzyme. By titrating the reduction in the extent of the burst of synthesis observed in a reaction incorporating dideoxythymidine monophosphate into poly(rA)-oligo(dT)18, the apparent equilibrium constant for dissociation of the reverse transcriptase-L-696,229 complex was estimated to be 400 nM.

Chronic B19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (HIV-1) is one cause of reversible anemia in this patient group. This report describes a case of concurrent HIV-1 and B19 parvovirus infection with pure red cell aplasia in which the anemia resolved with gammaglobulin treatment. When cultured in vitro with recombinant human stem cell factor, the red blood cell precursors from this patient demonstrated increases in both number and size, suggesting that simultaneous infection with B19 parvovirus and HIV-1 does not preclude a response to erythroid-acting growth factors. Although rare, persistent B19 parvovirus infection has become an increasingly recognized treatable cause of anemia in HIV-infected patients. Further in vitro and in vivo studies are required to determine whether cytokines such as stem cell factor have a consistent effect in these anemic states.

When the catalytically active, tyrosyl-phosphorylated form of insulin receptor was isolated from human placenta and treated with ADP, only partial dephosphorylation was observed. This observation suggests the existence of two distinct classes of phosphotyrosyl residues of the phosphorylated insulin receptor: one in which the phosphoryl groups undergo reversible transfer to ADP and one in which they do not. There were 8.8 +/- 2.2 phosphorylation sites per tetrameric insulin receptor, of which 2.4 +/- 0.5 were irreversible (mean +/- S.D., n = 6). The reversible sites were determined to be equivalent and noninteracting and to have an equilibrium constant for the transfer of a phosphoryl group from ATP to a site of reversible phosphorylation of 8.7. Surprisingly, both phosphorylation and dephosphorylation at the reversible sites were relatively insensitive to the presence of insulin. Since only minimal autophosphorylation of insulin receptor was detected in the absence of insulin, the results suggest that the incorporation of phosphate into the two to three irreversible phosphorylation sites is insulin dependent. Phosphorylation of the irreversible phosphorylation sites then renders the insulin receptor relatively insensitive to the continued presence of insulin and facilitates rapid reversible phosphorylation of a second group of tyrosyl residues. The dependence of the degree of phosphorylation of insulin receptor on the ATP:ADP ratio may provide a mechanism for modulating the cellular response to insulin.