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Background Transthyretin (TTR) amyloidosis is an underdiagnosed disease caused by destabilization of TTR due to pathogenic mutations or aging. Both pathogenic and protective mutations illuminate mechanisms of disease and potential interventions. AG10 is a selective, oral TTR stabilizer under development for transthyretin amyloidosis cardiomyopathy (ATTR-CM) that mimics a protective TTR mutation. Objectives This randomized, double-blind, placebo-controlled study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics of AG10 in ATTR-CM patients with symptomatic, chronic heart failure. Methods ATTR-CM, New York Heart Association functional class II to III subjects (n = 49, mutant or wild-type) were randomized 1:1:1 to AG10 400 mg, AG10 800 mg, or placebo twice daily for 28 days. Safety and tolerability were assessed by clinical and laboratory criteria. AG10 plasma levels were measured. TTR stability was assessed by changes in serum TTR, and 2 established ex vivo assays (fluorescent probe exclusion and Western blot). Results AG10 treatment was well-tolerated, achieved target plasma concentrations and demonstrated near-complete stabilization of TTR. TTR stabilization was more complete and less variable at the higher dose with stabilization by fluorescent probe exclusion of 92 ± 10% (mean ± SD) at trough and 96 ± 9% at peak (both p Conclusions AG10 has the potential to be a safe and effective treatment for patients with ATTR-CM. A phase 3 trial is ongoing. (Study of AG10 in Amyloid Cardiomyopathy; NCT03458130)

AG10 is a novel, potent, and selective oral transthyretin (TTR) stabilizer being developed to treat TTR amyloidosis (ATTR). This randomized, double‐blind, placebo‐controlled study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics (ex vivo stabilization) of orally administered AG10 in healthy adult volunteers. Both mutant and wild‐type ATTR are underdiagnosed diseases with limited therapeutic options. As TTR amyloidogenesis is initiated by dissociation of TTR tetramers destabilized due to inherited mutations or aging, AG10 is designed to treat the disease at its source. Four single and three multiple ascending dose levels of AG10 or matching placebo were orally administered. Safety and tolerability were assessed by vital signs, electrocardiogram, adverse events, and clinical laboratory tests. Pharmacokinetics were measured using a validated bioanalytical assay. Pharmacodynamics were assessed via three pharmacodynamic assays of TTR stabilization. AG10 was uniformly well tolerated, and no safety signals of clinical concern were observed. Pharmacokinetic observations included time to maximum concentration 90%) stabilization of TTR was observed across the entire dosing interval at steady state on the highest dose tested. Serum TTR levels, an in vivo reflection of TTR stabilization by AG10, increased from baseline following 12 days of dosing. AG10 appears to be safe and well tolerated in healthy adult volunteers and can completely stabilize TTR across the dosing interval, establishing clinical proof of concept. Based on these data, AG10 has the potential to be a safe and effective treatment for patients with either mutant or wild‐type ATTR.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While pathogenic TTR mutations (TTRm) destabilize TTR tetramers, the T119M variant stabilizes TTRm and prevents disease. A comparison of potency for leading TTR stabilizers in clinic and structural features important for effective TTR stabilization is lacking. Here, we found that molecular interactions reflected in better binding enthalpy may be critical for development of TTR stabilizers with improved potency and selectivity. Our studies provide mechanistic insights into the unique binding mode of the TTR stabilizer, AG10, which could be attributed to mimicking the stabilizing T119M variant. Because of the lack of animal models for ATTR-CM, we developed an in vivo system in dogs which proved appropriate for assessing the pharmacokinetics-pharmacodynamics profile of TTR stabilizers. In addition to stabilizing TTR, we hypothesize that optimizing the binding enthalpy could have implications for designing therapeutic agents for other amyloid diseases.

Introduction AG10 is a novel, potent and selective oral transthyretin (TTR) stabilizer under clinical development to treat TTR amyloidosis (ATTR). Both mutant and wild-type ATTR are under-diagnosed diseases with no FDA-approved therapies. As ATTR is caused by the destabilization of TTR tetramers due to inherited mutations or aging, AG10 is designed to treat the disease at its source. Hypothesis AG10 is safe and well tolerated at exposures reaching target therapeutic plasma concentrations, resulting in complete stabilization of TTR following repeat dosing in healthy volunteers (HV), and predicting clinical efficacy in treating both mutant and wild-type ATTR. Methods Single and multiple ascending oral doses of AG10 were administered in a double blind manner to 4 single and 3 multiple dose cohorts consisting of 8 HV per cohort randomized 3:1 (AG10: placebo). Safety and tolerability were assessed by vital signs, ECG, adverse events, and safety labs to monitor kidney and liver function, electrolytes, and hematology. Pharmacokinetics were measured using a validated bioanalytical assay. Pharmacodynamics (PD) were assessed via two established ex vivo assays for TTR stabilization, fluorescent probe exclusion and Western blot. Results Single oral doses of 50, 150, 300 and 800 mg, and multiple oral doses to steady state of 100, 300 and 800 mg q12h were well tolerated with a clean safety profile. AG10 was rapidly absorbed with t max max and AUC were dose-dependent, terminal t 1/2 was ∼25 hr, and there was little food effect (slightly increased T max , blunted C max , and 11% increase in AUC following a high fat meal). The PD profile, as assessed by either assay, demonstrated complete stabilization of TTR at C max following single doses of 300 mg or more, and >95% stabilization on average across the entire dosing interval of 800 mg q12h at steady state with low inter-subject variability (Figure). Conclusions AG10, a novel, potent and selective oral TTR stabilizer, is safe and well-tolerated following oral dosing in healthy volunteers and completely stabilizes TTR (>90%) across the dosing interval, establishing clinical proof of concept. Based on these data, AG10 has the potential to be a safe and effective treatment for patients with ATTR.

AG10 is a highly selective and potent oral transthyretin (TTR) stabilizer under development for TTR amyloidosis (ATTR). ATTR cardiomyopathy (ATTR-CM) is under-diagnosed with no FDA-approved therapies indicated for its treatment. Hypothesis: At doses investigated, AG10 achieves complete stabilization

Background: CLEC-2 signals through a cytosolic YXXL downstream of a triacidic amino acid sequence (hemITAM). Results: We show a critical role for the triacidic amino acid in phosphorylation of the cytosolic tyrosine and that CLEC-2 signals via Syk but not Zap-70. Conclusion: CLEC-2 signaling requires a cytosolic triacidic amino acid sequence and Syk. Significance: Proximal signaling events by hemITAM receptors are distinct to those in ITAM receptors., CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

Leptin therapy in human recombinant form has recently been used in HIV-associated lipodystrophy syndrome on experimental basis in some small short-term clinical trials. It has shown its beneficial effects only in hypoleptinemic HIV-infected patients by causing definite improvement in their insulin sensitivity, glucose tolerance, lipid status, and truncal obesity. Leptin prevents lipotoxicity and activates insulin signaling pathways through several postulated mechanisms. Central leptin insufficiency with peripheral hyperleptinemia has come out to be a significant contributor to the development of obesity and metabolic syndrome. In this article, we will review the basis of leptin therapy in HIV patients, with its promises. However, further larger clinical trials are needed to prove its long-term efficacy in the control of metabolic complications related to HIV therapy.

Disease development in human T-cell leukemia virus type 1 (HTLV-1)–infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication.

Whereas adaptive immunity has been extensively studied, very little is known about the innate immunity of the host to HTLV-I infection. HTLV-I-infected ATL patients have pronounced immunodeficiency associated with frequent opportunistic infections, and in these patients, concurrent infections with bacteria and/or parasites are known to increase risks of progression to ATL. The Toll-like receptor-4 (TLR4) activation in response to bacterial infection is essential for dendritic cell maturation and links the innate and adaptive immune responses. Recent reports indicate that TLR4 is targeted by viruses such as RSV, HCV, and MMTV. Here we report that HTLV-I has also evolved a protein that interferes with TLR4 signaling; p30 interacts with and inhibits the DNA binding and transcription activity of PU.1 resulting in the down-regulation of the TLR4 expression from the cell surface. Expression of p30 hampers the release of pro-inflammatory cytokines MCP-1, TNF-alpha, and IL-8 and stimulates release of anti-inflammatory IL-10 following stimulation of TLR4 in human macrophage. Finally, we found that p30 increases phosphorylation and inactivation of GSK3-beta a key step for IL-10 production. Our study suggests a novel function of p30, which may instigate immune tolerance by reducing activation of adaptive immunity in ATL patients.

Enzymes of the blood coagulation pathway enhance the inflammatory response leading to endothelial dysfunction, accounting, in part, for the vascular complications occurring in sepsis and cardiovascular disease. The responses of endothelial cell activation include induction of the expression of tissue factor (TF), a membrane glycoprotein that promotes thrombosis, and of E-selectin, a cell adhesion molecule that promotes inflammation. In this report, we demonstrate synergistic interactions between the coagulation factor Xa (fXa) and the proinflammatory cytokines TNF, IL-1beta, and CD40L, leading to enhanced expression of TF and E-selectin in endothelial cells. A detailed analysis of the molecular pathways that could account for this activity of fXa showed that fXa inhibited the cytokine-induced expression of dual specificity phosphatases, MAP kinase phosphatase-L, -4, -5, and -7, blocking a negative regulatory effect on c-Jun N-terminal kinase. The synergistic interaction between fXa and TNF was also involved in the inhibition of A20 and IkappaBalpha expression in the IkappaB kinase-NF-kappaB pathway. The data indicate that inhibition of negative regulatory signaling accounts for the amplification of cytokine-induced endothelial cell activation by fXa.

In immortal cells, the existence of a mechanism for the maintenance of telomere length is critical. In most cases this is achieved by the reactivation of telomerase, a cellular reverse transcriptase that prevents telomere shortening. Here we report that the telomerase gene (hTERT) promoter is up-regulated during transmission of human T-cell lymphotropic virus type-I (HTLV-I) to primary T cells in vitro and in ex vivo adult T-cell leukemia/lymphoma (ATLL) samples, but not asymptomatic carriers. Although Tax impaired induction of human telomerase reverse transcriptase (hTERT) mRNA in response to mitogenic stimulation, transduction of Tax into primary lymphocytes was sufficient to activate and maintain telomerase expression and telomere length when cultured in the absence of any exogenous stimulation. Transient transfection assays revealed that Tax stimulates the hTERT promoter through the nuclear factor κB (NF-κB) pathway. Consistently, Tax mutants inactive for NF-κB activation could not activate the hTERT or sustain telomere length in transduced primary lymphocytes. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays suggested that an increased binding of c-Myc and Sp1 is involved in the NF-κB–mediated activation of the hTERT promoter. This study establishes the role of Tax in regulation of telomerase expression, which may cooperate with other functions of Tax to promote HTLV-I–associated adult T-cell leukemia.

Using N,N-dialkylated benzamidines as the novel P4 motifs, we have designed and synthesized a class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent and selective fXa inhibitors with significantly improved hydrophilicity and in vitro anticoagulant activity. These benzamidine-P4 fXa inhibitors have displayed excellent oral bioavailability and long half-life.

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)or =2 nM) with improved in vitro anticoagulant activity (2xTGor =1 microM) and respectable pharmacokinetic properties have been discovered.

Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors. Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly potent in vitro, and show improved activity in human plasma-based thrombin generation assay.

Hypothyroidism presenting as recurrent hypokalemic paralysis is rare in the literature. This transient and episodic neurological condition is commonly associated with thyrotoxicosis. We report a case of young female admitted with recurrent paralytic attacks since last 1 year. She had no symptom of hypothyroidism. She had weakness of all four limbs, delayed relaxation of ankle jerks, and normal higher mental function. There was no enlargement of thyroid. Serum potassium level ranged from 1.6 to 3.2 meq/L during attack with high serum creatine phosphokinase level. Electromyography was normal. The patient was diagnosed having chronic thyroiditis with high thyroid-stimulating hormone and thyroid-related antibodies. Follow up shows satisfactory result with thyroxine replacement. It is an extremely rare and unusual presentation of hypothyroidism, probably the fourth reported case of hypothyroidism with hypokalemic paralysis, to the best of our knowledge.

A total of 28 consecutive patients suffering from alcoholic liver disease who were admitted to the Department of Medicine, Nilratan Sircar Medical College and Hospital, Kolkata, from January 2009 to August 2010, were enrolled in this study. Patients in whom liver biopsy was contradicted because of co-morbid conditions like coagulopathy, tense ascites and hepatic space occupying lesions, or those who did not consent for biopsy were excluded from the study.

To overcome the low bioavailability of our substituted acrylamide P1 benzamidine factor Xa inhibitors reported previously, neutral and less basic groups were used to replace the benzamidine. As a result, a series of P1 aminoisoquinoline substituted acrylamide Xa inhibitors was identified to be potent, selective, and orally bioavailable. Modification of P4 moiety of these compounds further improved their pharmacokinetic properties.

Monoamidine FXa inhibitors 3 were designed and synthesized. SAR studies and molecular modeling led to the design of conformationally constrained diaryl ethers 4 and 5, as well as benzopyrrolidinone 7 as potent FXa inhibitors. The monoamidines show high efficacy in a DVT model, but lack desirable oral bioavailability. The benzopyrrolidinone-based aminoisoquinolines 8 do not show significant improvement in oral bioavailability.

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX-glutathione S-transferase (RafX-GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(gamma-OR)-Gly-Arg-pNA.HCl], S2765 [Z-D-Arg-Gly-Arg-pNA.HCl] and Spectrozyme Xa [MeO-CO-D-CHG-Gly-Arg-pNA.AcOH]). Michaelis constants (K(m)) for the rabbit protein were 187, 72 and 69 microM, respectively, and for the human analog, 255, 63 and 135 microM, respectively. Comparing the extent of substrate turnover (V(max)) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.

A series of potent and selective factor Xa inhibitors was synthesized using various readily available amino acids as central templates. The most potent compound displays IC50 of 3 nM.

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p′-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the “tenase” complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus associated with the lymphoproliferative disease adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disorder tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Replication of HTLV-1 is under the control of two major trans -acting proteins, Tax and Rex. Previous studies suggested that Tax activates transcription from the viral long terminal repeat (LTR) through recruitment of cellular CREB and transcriptional coactivators. Other studies reported that Rex acts posttranscriptionally and allows the cytoplasmic export of unspliced or incompletely spliced viral mRNAs carrying gag/pol and env only. As opposed to HIV's Rev-responsive element (RRE), the Rex-responsive element (RxRE) is present in all viral mRNAs in HTLV-1. However, based on indirect observations, it is believed that nuclear export and expression of the doubly spliced tax/rex RNA are Rex independent. In this study, we demonstrate that Rex does stimulate Tax expression, through nuclear-cytoplasmic export of the tax / rex RNA, even though a Rex-independent basal export mechanism exists. This effect was dependent upon the RxRE element and the RNA-binding activity of Rex. In addition, Rex-mediated export of tax / rex RNA was CRM1 dependent and inhibited by leptomycin B treatment. RNA immunoprecipitation (RNA-IP) experiments confirmed Rex binding to the tax / rex RNA in both transfected cells with HTLV-1 molecular clones and HTLV-1-infected T cells. Since both Rex and p30 interact with the tax/rex RNA and with one another, this may offer a temporal and dynamic regulation of HTLV-1 replication. Our results shed light on HTLV-1 replication and reveal a more complex regulatory network than previously anticipated.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 μM, respectively) and Fce receptor 1-mediated basophil degranulation (IC50 0.15 μM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 μM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

Heparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and γ-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 × 10−10 m ). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.

Anthranilamide-based benzamidine compound 4 and its N-substituted analogs were designed and examined as factor Xa inhibitors using substituted benzamidines as unconventional S4 binding element. A group of N,N-dialkylbenzamidines (11, 17 and 24) have been discovered as potent factor Xa inhibitors with strong anticoagulant activity and promising oral PK profiles.

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.

1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. Differential keratinolytic substrate specificity among these two microbes are probable due to a difference in disulfide reduction capacity.

Human T-cell lymphotrophic virus type I Rex and p30 are both RNA binding regulatory proteins. Rex is a protein that interacts with a responsive element and stimulates nuclear export of incompletely spliced viral RNAs thereby increasing production of virus particles. In contrast, p30 is involved in the nuclear retention of the tax/rex mRNA leading to inhibition of virus expression and possible establishment of viral latency. How these two proteins, with apparent opposite functions, integrate in the viral replication cycle is unknown. Here, we demonstrate that Rex and p30 form ribonucleoprotein ternary complexes onto specific viral mRNA. Our results explain the selective nuclear retention of tax/rex but not other viral mRNAs by p30. Whereas p30 suppresses Rex expression, it did not affect Rex-mediated nuclear export of RNA containing the Rex response element. In contrast, Rex was able to counteract p30-mediated suppression of viral expression and restore cytoplasmic tax/rex mRNA and Tax protein expression. Together, our data demonstrate a complex regulatory mechanism of antagonizing post-transcriptional regulators evolved by human T-cell lymphotrophic virus type I to allow a vigilant control of viral gene expression.

Genotoxicity of crotonaldehyde was evaluated by employing bone marrow and spermatocyte chromosomal aberration and dominant lethal mutation assays in Swiss albino mice. For bone marrow chromosomal aberration assay, animals were treated with 0.2 ml olive oil containing 8, 16 and 32 μl kg b.w. of crotonaldehyde for 6, 12 and 24 h through single intraperitoneal injection. Treatment induced dose-dependent and statistically significant decrease in mitotic index and increase in chromosome aberrations per cell (CAs/cell excluding gaps and stickiness and pulverizations) and percent aberrant metaphase (excluding gaps) in the bone marrow cells at 6, 12 and 24 h after treatment. For spermatocyte chromosomal aberration assay, male mice were treated with 8, 16 and 32 μl/kg b.w. of crotonaldehyde for 24 h through single intraperitoneal injection. The percentage of the induced chromosome aberrations in diakinesis-metaphase-I cells showed dose-dependent increase. For dominant lethal mutation assay, adult male mice were injected intraperitoneally with 0.2 ml olive oil containing crotonaldehyde at the rate of 8, 16 and 32 μl/kg b.w. for 5 consecutive days. The negative control animals received 0.2 ml olive oil as above. In the dominant lethal mutation assay after the last dose, the treated males were allowed to mate with untreated virgin females. The mating continued for 5 consecutive weeks. At pregnancy days 14–16, the females were killed and uterine contents were examined for live and dead implants. Treatment of mice resulted in statistically significant decrease in the fertility indices and total number of implants per female. Statistically significant decrease in the number of live implants per female and increase in the number of dead implants per female were recorded in females mated with males during 8–14 and 15–21 and 22–28 days post-treatment mating. Crotonaldehyde treatment of males induced appreciably higher frequencies of dominant lethal mutation during 8–14, 15–21 and 22–28 days post-treatment mating periods. Percent dominant lethal mutation increased concomitantly with the dose. Dominant lethality was maximum in females mated with males treated with 5 × 32 μl/kg b.w. during the 15–21 days post-treatment mating. The overall result suggests a positive dose–response relationship between treatment and induction of chromosomal aberrations in the somatic and germ cells and dominant lethal mutation in the germ cells.

Human T-cell leukemia virus type I is the etiological agent of adult T-cell leukemia/lymphoma, an aggressive and fatal lymphoproliferative malignancy. The virus has evolved strategies to escape immune clearance by remaining latent in most infected cells in vivo. We demonstrated previously that virally encoded p30 protein is a potent post-transcriptional inhibitor of virus replication (Nicot, C., Dundr, M., Johnson, J. M., Fullen, J. R., Alonzo, N., Fukumoto, R., Princler, G. L., Derse, D., Misteli, T., and Franchini, G. (2004) Nat. Med. 10, 197-201). p30 is unable to shuttle out of the nucleus in heterokaryon assays, suggesting the existence of specific retention signals. Because suppression of virus replication relies on nuclear retention of the tax/rex mRNA by p30, determining the retention features of p30 will offer hints to break latency in infected cells and insights into new therapeutic approaches. In this study, we used live cell imaging technologies to study the kinetics of p30 and to delineate its retention signals and their function in virus replication. Notably, this is the first study to identify p30 nucleolar retention domains. Using mutants of p30 that localized in different cellular compartments, we show that post-transcriptional control of virus replication by p30 occurs in the nucleoplasm. We further demonstrate that p30 nuclear/nucleolar retention is dependent upon de novo RNA transcripts and interactions with components of the ribosomal machinery.

Drug-induced QT prolongation arising from drugs' blocking of hERG channel activity presents significant challenges in drug development. Many, but not all, of our benzamidine-containing factor Xa inhibitors were found to have high hERG binding propensity. However, incorporation of a carboxylic acid group into these benzamidine molecules generally leads to hERG inactive compounds regardless where the carboxyl group is tethered within the molecules. The inhibitory effect of a carboxylic acid group on hERG binding has also been observed in many series of diverse structural scaffolds (including non-amidines). These findings suggest that the negatively charged carboxylate group causes unfavorable interaction within hERG channel binding cavity by electrostatic interaction.

The antiviral thymidine analog azidothymidine (AZT) is used to treat several virus-associated human cancers. However, to date the mechanism of AZT action remains unclear and thus, reasons for treatment failure are unknown. Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of poor prognosis. Here, we report that enduring AZT treatment of T-cell leukemia virus I–infected cells, in vitro and in vivo in ATL patients, results in inhibition of telomerase activity, progressive telomere shortening, and increased p14ARF expression. In turn, this elicits stabilization and reactivation of the tumor suppressor p53-dependent transcription, increased expression of the cyclin-dependent kinase inhibitor p21Waf1, and accumulation of p27kip1, thereby inducing cellular senescence and tumor cell death. While ATL patients carrying a wild-type p53 enter remission following treatment with AZT, those with a mutated p53 did not respond, and patients' disease relapse was associated with the selection of a tumor clone carrying mutated inactive p53.

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.

A class of N,N-dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines has been discovered as potent factor Xa inhibitors with desirable in vitro and in vivo anticoagulant activity, but with low oral bioavailability. The 5-chloroindole and 6-chlorobenzo[b]thiophene groups are optimal as the factor Xa S1 binding elements. The strategy of incorporating a side chain on the piperazine nucleus to enhance binding affinity has been examined.

Parallel synthesis and iterative optimization led to the discovery of a series of potent and specific factor Xa inhibitors demonstrating excellent in vitro activity with promising pharmacokinetics.

Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure–activity relationship studies around 2 have led to a series of potent factor Xa inhibitors which are highly active in the human plasma-based thrombin generation assay with 2XTG values less than 1 μM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein thrombosis model, and also exhibits good oral bioavailability and a long half life in rats.

A series of novel transition state factor Xa inhibitors containing a variety of lactam ring systems as central templates was synthesized in an expedient manner and allowed for a great deal of structural variability. Among them, the piperazinone-based inhibitors were found to be not only active against factor Xa but also selective over thrombin. Optimization of the P4 moiety yielded several potent compounds with IC(50) below 1 nM against factor Xa.

A series of benzoxazinone derivatives was designed and synthesized as factor Xa inhibitors. We demonstrated that the naphthyl moiety in the aniline-based compounds 1 and 2 can be replaced with benzene-fused heterobicycles and biaryls to give factor Xa inhibitors with improved trypsin selectivity. The P4 modifications lead to monoamidines which are moderately active. The benzoxazinones 41-45 are potent against factor Xa, retain the improved trypsin selectivity of the corresponding aniline-based compounds, and show strong antithrombotic effect dose responsively.

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3 , a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency ( 14 , IC 50 =0.028 μM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.

Based on DuPont Pharmaceuticals' monobenzamidine lead structure SN429, we have designed the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as a novel series of non-basic factor Xa inhibitors. We have discovered that the displacement of the benzamidine moiety with substituted 2-naphthyl structures not only results in highly potent factor Xa inhibitors, but also significantly increases their enzyme specificity and oral bioavailability.

A series of glycolic and mandelic acid derivatives was synthesized and investigated for their factor Xa inhibitory activity. These analogues are highly potent and selective inhibitors against fXa. In a rabbit deep vein thrombosis model, compound 26 showed significant antithrombotic effects (81% inhibition of thrombus formation) at 1.1 microM plasma concentration following intravenous administration.

Recently, peptidylketothiazoles have been shown to be potent inhibitors of proteases, but the details of the interaction have not yet been studied. In the work presented here, the interaction of factor Xa, a coagulation protease, with the transition state inhibitor BnSO(2)-D-Arg-Gly-Arg-ketothiazole (C921-78) is characterized. C921-78 is a tight and selective inhibitor of the coagulation protease factor Xa (K(d) = 14 pM). The hydrolytic activity of factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-limiting step of the bimolecular combination of inhibitor and enzyme was competitive with the substrate. Conversely, the inhibitor could be displaced from the active site of the enzyme after exposure of the preformed complex to an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921-78-factor Xa complex resulted in a 60% increase in the magnitude of the fluorescence emission spectrum. Rapid mixing of the enzyme and inhibitor produces a monophasic fluorescence increase, compatible with spectral transition in a single step. The rate constant for this reaction increased hyperbolically with the concentration of C921-78, but the amplitude remained constant. These results are consistent with the initial formation of an enzyme-inhibitor complex (EI), followed by a unimolecular conversion of EI to EI linked to a spectral transition. The rate constants of the isomerization provide an estimate of 300000-fold stabilization. Thus, the inhibition of factor Xa by C921-78 follows a mechanism similar to that described classically for slow tight binding inhibitors. However, the two steps of the reaction cannot be kinetically separated by the rapid equilibrium assumption, and therefore, the formation of EI is partially rate-limiting, too. The driving energy for the unusually fast isomerization step may result from the highly favorable interactions of the inhibitor in the primary binding site.

Five new quinone pigments have been discovered from the fermentation broth of Aspergillus, Humicola and Botryotrichum species isolated from different soil samples. These compounds inhibit serine proteases of the coagulation pathway. Their structures which differ in the identity and position of a 5-carbon side chain on the indole moiety have been elucidated based on NMR and FAB-MS experiments.

Abstract 3532 B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL). PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.

Abstract 2764 The heterogeneity and severity of certain autoimmune diseases and B cell malignancies warrant simultaneous targeting of multiple disease-relevant pathways. Simultaneous inhibition of Syk and JAK represents such a strategy, and may have several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for alternate disease mechanisms, and the potential that an overall lower level suppression of multiple targets may be sufficient to modulate disease activity. To this end, we provide an update on the pre-clinical development of our lead dual Syk/JAK inhibitor; PRT062070. Cellular assays demonstrate an impressive selectivity profile, with potency against Syk, JAK1, and JAK3 primarily maintained in whole blood assays. B cell antigen-receptor mediated cellular activation and FcεRI-mediated basophil degranulation were inhibited in whole blood by PRT062070 with Syk IC50's of 0.1μM and 0.23μM, respectively. PRT062070 preferentially inhibited JAK1 and JAK3 dependent cytokine mediated signaling and functional responses in various cell types. IL2 mediated STAT5 Y694 was inhibited with an IC50 of 0.27μM, while IL4 mediated signaling to STAT6 Y641 and functional responses in B cells and monocytes, namely CD69, CD25, and CD23 up-regulation, were inhibited with IC50's within the range of 0.11μM to 0.57μM. PRT062070 also demonstrated potency in suppressing FcγR mediated neutrophil oxidative burst. Conversely, PRT062070 did not inhibit T cell antigen receptor or PMA-mediated signaling to ERK Y204, or T cell receptor mediated Lck activity as measured by ZAP70 Y319, providing evidence for selectivity of action. At 4uM (the highest concentration tested) only 20% inhibition against GM-CSF mediated STAT5 phophorylation in monocytes was observed, suggesting limited inhibition of JAK2. In tumor cell viability assays, several non-Hodgkins lymphoma cell lines demonstrated enhanced sensitivity to dual Syk/JAK inhibition relative to JAK inhibition alone. Following oral dosing in rodents, PRT062070 suppressed inflammation in the mouse collagen antibody induced arthritis model, prevented splenomegaly in mice stimulated in vivo with BCR specific antibody, and diminished progression of a B cell non-Hodgkins lymphoma cell line in xenograft mice. Using the rat collagen induced arthritis treatment model, we report a dose-dependent inhibition of inflammation with complete suppression of disease progression at 0.3μM. The advantages of dually targeting Syk and JAK for inflammation and B cell cancers will be discussed, as well as plans for initial human studies. Disclosures: Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. DeGuzman:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Lee:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Kamen:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Baker:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Hollenbach:Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.

The plasma proteinase inhibitor corresponding to alpha-1-proteinase inhibitor (alpha 1Pl) in humans was isolated from sheep plasma. Ovine alpha 1Pl is of higher molecular weight (62,000 daltons) than is human alpha 1Pl, is resistant to chemical oxidation by N-chlorosuccinimide, and has poor elastase-inactivating power compared with the corresponding inhibitor in humans. However, ovine alpha 1Pl is a potent trypsin inhibitor. Despite the differences indicated above, a partial homology (22 to 35%) exists between human and sheep alpha 1Pl, at least as analyzed through the first 20 residues of the sheep inhibitor. The weak elastase-inhibitory capacity of sheep alpha 1Pl is paralleled by the low content of elastase in the sheep neutrophil granule. These important differences between sheep and human neutrophils and plasma proteinase inhibitors should be borne in mind in designing experiments related to proteolytically mediated lung injury in the former species.