Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430.

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.

BCX4430, a novel nucleoside analog, effectively treats yellow fever in a Hamster model.

No effective antiviral therapies are currently available to treat disease after infection with yellow fever virus (YFV). A Syrian golden hamster model of yellow fever (YF) was used to characterize the effect of treatment with BCX4430, a novel adenosine nucleoside analog. Significant improvement in survival was observed after treatment with BCX4430 at 4 mg/kg of body weight per day dosed intraperitoneally (i.p.) twice daily (BID). Treatment with BCX4430 at 12.5 mg/kg/day administered i.p. BID for 7 days offered complete protection from mortality and also resulted in significant improvement of other YF disease parameters, including weight loss, serum alanine aminotransferase levels (6 days postinfection [dpi]), and viremia (4 dpi). In uninfected hamsters, BCX4430 at 200 mg/kg/day administered i.p. BID for 7 days was well tolerated and did not result in mortality or weight loss, suggesting a potentially wide therapeutic index. Treatment with BCX4430 at 12 mg/kg/day i.p. remained effective when administered once daily and for only 4 days. Moreover, BCX4430 dosed at 200 mg/kg/day i.p. BID for 7 days effectively treated YF, even when treatment was delayed up to 4 days after virus challenge, corresponding with peak viral titers in the liver and serum. BCX4430 treatment did not preclude a protective antibody response, as higher neutralizing antibody (nAb) concentrations corresponded with increasing delays of treatment initiation, and greater nAb responses resulted in the protection of animals from a secondary challenge with YFV. In summary, BCX4430 is highly active in a hamster model of YF, even when treatment is initiated at the peak of viral replication.

Synthesis and biological evaluation of Ribo 7-N/O/S pyrimidine 9-deaza C-nucleoside analogs as new antiviral agents for inhibiting HCV RNA-dependent RNA polymerases.

Hepatitis C infection is caused by the bloodborne pathogen hepatitis C virus (HCV) and can lead to serious liver diseases and, ultimately, death if the treatment is ineffective. This work reports the synthesis and preclinical evaluation of 7 novel 9-O/N/S pyrimidine nucleosides, including compound 12, the triphosphate of known compound 7b. The nucleosides are 9-deaza modifications of adenosine and guanosine with ß-2'-C-methyl substituent on the ribose. Within this series of compounds, a 9-deaza furopyrimidine analog of adenosine, compound 7b, showed high anti-HCV activity in vitro, good stability, low toxicity, and low genotoxicity when administrated in low doses, and an adequate pharmacokinetics profile. An improved synthesis of compound 7b compared to a previous study is also reported. Compound 12 was synthesized as a control to verify phosphorylation of 7b occurred in vivo.

In vitro efficacy of forodesine and nelarabine (ara-G) in pediatric leukemia.

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Synthesis of novel azasugar-containing 2'ß-C-Me 9-deaza nucleosides as potential anti-hepatitis C virus agents.

As a part of our ongoing discovery efforts exploring azasugar as agents for treating various unmet medical needs, we prepared analogs of azasugar as potential anti-hepatitis C virus (HCV) agents. Herein we describe the synthesis of novel 2'ß-C-Me 9-deazanucleoside azasugar analogs.

Phase 2 and pharmacodynamic study of oral forodesine in patients with advanced, fludarabine-treated chronic lymphocytic leukemia.

Forodesine is a new and potent purine nucleoside phosphorylase (PNP) inhibitor. Patients with chronic lymphocytic leukemia (CLL) with primary resistance to fludarabine-based therapy or with progressive disease were eligible for oral forodesine (200 mg/d) for up to 24 weeks. Eight patients with median lymphocyte count of 35.9 x 10(9)/L and median serum beta2 microglobulin level of 6.45 mg/L were treated. Six had Rai stage III to IV and were previously heavily treated (median prior therapy = 5). Two had transient decrease in lymphocyte count to normal, whereas in 5, disease progressed. Adverse events were mild. Steady-state level of forodesine ranged from 200 to 1300 nM and did not reach desired 2 microM level. PNP inhibition ranged from 57% to 89% and steady-state 2'-deoxyguanosine (dGuo) concentration median was 1.8 microM. Intracellular deoxyguanosine triphosphate (dGTP) increase was very modest, from median of 6 microM to 10 microM. Compared with in vivo, in vitro incubations of CLL lymphocytes with 10 or 20 microM dGuo and forodesine (2 microM) resulted in accumulation of higher levels of dGTP (40-250 microM) which resulted in increase in apoptosis. Forodesine has biologic activity in CLL; pharmacodynamic parameters suggest that an alternate dosing schedule and/or higher doses to achieve greater intracellular dGTP may be beneficial in this patient population.

Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Forodesine has high antitumor activity in chronic lymphocytic leukemia and activates p53-independent mitochondrial apoptosis by induction of p73 and BIM.

Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5(+) B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.

Design, parallel synthesis, and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores.

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF x FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF x FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC(50) value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF x FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure-activity relationship.

Correction to: Treatment duration with immune-based therapies in Cancer: an enigma.

Following publication of the original article [1], the authors reported an error in the typesetting of their article. The first section of the main text was mistakenly included in the abstract.

Treatment duration with immune-based therapies in Cancer: an enigma.

Unlike chemotherapy treatments that target the tumor itself (rather nonspecifically), immune-based therapies attempt to harness the power of an individual patient's immune system to combat cancer. Similar to chemotherapeutic agents, the dosage and Administration section of labeling for all five currently approved PD-1/PD-L1 inhibitors (immunotherapy) recommends duration of treatment until disease progression or unacceptable toxicity. Overactivation or constitutive activation of the immune system with immune based therapies can lead to T-cell exhaustion and activation induced cell death (AICD) in T- and B-cells. Examples of immune exhaustion and T-cell depletion is noted in preclinical and clinical studies. Overactivation or constitutive activation leading to Immune exhaustion is a real phenomenon and of profound concern as immune cells are the true arsenal for control of the tumor growth. Designing trials rigorously to address the optimum treatment duration with immune based therapies is critical. By addressing this concern now, not only we may improve patient outcomes, but also gather a deeper understanding of the role and mechanisms of the immune system in the control of tumor growth.Chemotherapy and immune-based therapies provide antitumor effects through completely different mechanisms. Chemotherapeutic agents are cytotoxic in that they directly inhibit basic cellular mechanisms, killing both malignant and nonmalignant cells (hopefully with a preference for malignant cells), while immune based therapies wake-up the host immune system to recognize malignant cells and eliminate them.While there is a burgeoning excitement surrounding development of immune based therapies for the treatment of cancer, the optimal duration for these therapies need to be explored with equal fervor. Dosing for chemotherapy has been determined over years through large-scale prospective randomized trials to pinpoint the dose which maximizes therapeutic effect while minimizing side effects. Also, due to the mechanism of chemotherapeutic action, the duration of treatment with these agents is generally until disease progression or patient intolerance. However, experience with immune based therapies is limited, with current dosing and duration guidelines based primarily on initial trials required for approval of the agents. Since immune based therapies work by activating the body's own immune system, there is concern that overactivation or constitutive activation of the immune system may lead to immune exhaustion and depletion of effector T-cells thereby causing decreased anti-tumor effects and possible allowing for tumor progression.Similar to chemotherapeutic agents, the Dosage and Administration section of labeling for all five currently approved PD-1/PD-L1 inhibitors recommends duration of treatment until disease progression or unacceptable toxicity. However, since immune based therapies work with a completely different mechanism compared to chemotherapy, using the same therapy duration may not be the optimal approach.In exploring treatment duration with immune based therapies, we need to answer the following: (1) does indefinite treatment with immune based therapies exhaust the immune system counteracting its own mechanism of action leading to tumor progression and (2) how can clinical trials be designed to identify the optimal duration of immune-based therapy that prevents immune cell exhaustion but supports anti-tumor immunity.

Anti-influenza virus activity of peramivir in mice with single intramuscular injection.

In the event of an influenza outbreak, antivirals including the neuraminidase (NA) inhibitors, peramivir, oseltamivir, and zanamivir may provide valuable benefit when vaccine production is delayed, limited, or cannot be used. Here we demonstrate the efficacy of a single intramuscular injection of peramivir in the mouse influenza model. Peramivir potently inhibits the neuraminidase enzyme N9 from H1N9 virus in vitro with a 50% inhibitory concentration (IC(50)) of 1.3+/-0.4 nM. On-site dissociation studies indicate that peramivir remains tightly bound to N9 NA (t(1/2)>24h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme (t(1/2)=1.25 h). A single intramuscular injection of peramivir (10mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H1N1, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N1 NA enzymes similar to that observed for N9 enzyme. Additional efficacy studies indicate that a single injection of peramivir (2-20mg/kg) was comparable to a q.d.x 5 day course of orally administered oseltamivir (2-20mg/kg/day) in preventing lethality in H3N2 and H1N1 influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak.

The antithrombotic and anti-inflammatory effects of BCX-3607, a small molecule tissue factor/factor VIIa inhibitor.

Tissue factor (TF) is a transmembrane glycoprotein that binds its zymogen cofactor, Factor VIIa (FVIIa) on the cell surface. Together (TF/FVIIa) they activate Factor X (FX) and Factor IX (FIX) and start the extrinsic pathway of blood coagulation. As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and deep vein thrombosis (DVT). In addition to its function as an initiator of coagulation, TF/FVIIa plays an important role in inflammation. Expression of TF on the cell surface and its appearance as a soluble molecule are characteristic features of acute and chronic inflammation in conditions such as sepsis and atherosclerosis. Here we demonstrate that BCX-3607, a small molecule potent inhibitor of TF/FVIIa, reduces thrombus weight in an animal model of DVT. BCX-3607 also decreases the level of interleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia. Additionally, in vitro studies indicate that BCX-3607 blocks the generation of TF/FVIIa-induced IL-8 mRNA in human keratinocytes and reduces the TF/FVIIa-mediated generation of IL-6 and IL-8 in human umbilical vein endothelial cells (HUVEC). Therefore, BCX-3607 might block the TF/FVIIa-mediated coagulation and inflammation associated with pathological conditions.

An efficient synthesis of acyclic N7- and N9-adenine nucleosides via alkylation with secondary carbon electrophiles to introduce versatile functional groups at the C-1 position of acyclic moiety.

The introduction of versatile functional groups, allyl and ester, at the C-1 position of the acyclic chain in acyclic adenine nucleosides was achieved for the first time directly by alkylation of adenine and N6-potected adenine. Thus, the C-1'-substituted N9-adenine acyclic nucleoside, adenine-9-yl-pent-4-enoic acid ethyl ester (11), was prepared by direct alkylation of adenine with 2-bromopent-4-enoic acid ethyl ester (6), while the corresponding N7-regioisomer, 2-[6-(dimethylaminomethyleneamino)-purin-7-yl]-pent-4-enoic acid ethyl ester (10), was obtained in one step by the coupling of N, N-dimethyl-N'- (9H-purin-6-yl)-formamidine (9) with 2-bromopent-4-enoic acid ethyl ester (6). The functional groups, ester and allyl, were converted to the desired hydroxymethyl and hydroxyethyl groups, and subsequently to phosphonomethyl derivatives and corresponding pyrophosphorylphosphonates.

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents.

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.

Synthesis of N6-substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives as possible antiviral agents.

A number of N6-substituted 9-[3-(phosphonomethoxy)propyl]adenine derivatives having hydroxymethyl at C-1' position were prepared from the appropriate 6-chloroadenine derivative. The syntheses of the corresponding prodrugs of these compounds are also reported. These compounds showed poor activity against HCV in replicon assay.

Purine nucleoside phosphorylase inhibitors in T-cell malignancies.

Purine nucleoside phosphorylase (PNP)-deficient children exhibit profound impairment in the T-cell component of their immune systems, but have normal B-cell function. This rare condition provides a model for the development of specific inhibitors of PNP, which should enable selective suppression of T-cell function that may be useful in the treatment of T-cell-mediated diseases. BCX-1777 (BioCryst Pharmaceuticals Inc) is a rationally designed, potent transition-state analog inhibitor of PNP. This review provides a summary of in vitro and in vivo inhibition studies of T-cells by BCX-1777, and the role in this process of plasma deoxyguanosine (dGuo) and intracellular deoxyguanosine triphosphate (dGTP). Preliminary data from a phase I clinical trial of BCX-1777 in patients with T-cell malignancy demonstrated antileukemic activity which can be correlated to an increase in plasma dGuo and intracellular dGTP. This is consistent with results observed in cell cultures, animal studies and PNP-deficient patients. Clinical trials with BCX-1777 have demonstrated that inhibition of PNP leads to T-cell-selective suppression, confirming PNP to be a promising target for the treatment of T-cell-mediated diseases.

Synthesis of a potent transition-state inhibitor of 5'-deoxy-5'-methylthioadenosine phosphorylase.

Human 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) participates in the purine salvage pathway to generate adenine and methylthioribose-1-phosphate, which in turn is converted into adenine nucleotides and methionine. Hence, inhibition of MTA phosphorylase may be an effective target in the design of potential antiproliferative agents. Presented herein is the synthesis of 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-methylsulfanylmethylpyrrolidin-3,4-diol (1), a potent inhibitor of MTAP.

Syntheses and neuraminidase inhibitory activity of multisubstituted cyclopentane amide derivatives.

In further studies aimed toward identifying effective and safe inhibitors of influenza neuraminidases, we synthesized a series of multisubstituted cyclopentane amide derivatives. Amides prepared were 14 examples of N-substituted alkyl or aralkyl types from primary amines, 13 examples of the N,N-disubstituted alkyl, aralkyl, or substituted-alkyl type from secondary amines, and 12 examples from cycloaliphatic or substituted cycloaliphatic secondary amines. These compounds bearing two chiral centers, at position-1 in the ring and position-1' in the side chain attached at position 3, were tested for their ability to inhibit A and B forms of influenza neuraminidase. The 1-ethylpropylamide, diethylamide, dipropylamide, and 4-morpholinylamide showed very good inhibitory activity (IC(50) = 0.015-0.080 microM) vs the neuraminidase A form, but modest activity (IC(50) = 3.0-9.2 microM) vs the neuraminidase B form. Since the parent amides bear two chiral centers (C-1 and C-1'), three of the better inhibitors were tested at higher levels of diastereomeric purity. The diastereomers corresponding to the active forms of the 1-(ethyl)propylamide, the diethylamide, and the dipropylamide (all of the same configuration at the C-1' chiral center), and the diastereomer of the diethylamide representing the active form at both C-1' and C-1 were isolated or synthesized from precursors that were isolated as diastereomers. These diastereomers showed some improvement in neuraminidase inhibition over the parent diastereomeric mixtures. 1-Carboxy-1-hydroxy derivatives of the best active compounds, the diethylamide and the dipropylamide, were also prepared. These compounds were not as active as the compounds without the 1-hydroxy group. In an in vivo study, the C-1' active isomer of the diethylamide from the 1-carboxy series was tested in influenza-infected mice by oral and intranasal administration and found to be very effective only intranasally in preventing weight loss at doses as low as 0.1 (mg/kg)/day.

Mechanism of inhibition of T-acute lymphoblastic leukemia cells by PNP inhibitor--BCX-1777.

Purine nucleoside phosphorylase (PNP) deficiency in humans produces a relatively selective depletion of T cells. BCX-1777 is a potent inhibitor of PNP. BCX-1777 in the presence of deoxyguanosine (dGuo) inhibits the proliferation of CEM-SS [T-acute lymphoblastic leukemia (T-ALL)] cells with an IC(50)=0.015 microM. This inhibition by BCX-1777 and dGuo is accompanied by elevation of dGTP (154-fold) and dATP (8-fold). Deoxycytidine (dCyt) completely and lamivudine (3TC) partially reverse this inhibition caused by BCX-1777 and dGuo. dNTP analysis of these samples indicates that, in the presence of dCyt, where complete reversal of inhibition is observed, dGTP and dATP pools revert back to the control levels. In samples containing 3TC, where partial reversal of inhibition was observed, dGTP decreased from 154-fold to 38-fold and dATP levels further increased from 8-fold to 30-fold compared to the control sample. In CEM-SS cells, inhibition of proliferation by BCX-1777 and dGuo is not due to accumulation of dATP because in the presence of 3TC, where reversal of inhibition is observed, dATP levels are further increased. These studies clearly indicate that inhibition of T cells is due to accumulation of dGTP resulting in cell death with characteristics of apoptosis. The half-life of dGTP in CEM-SS cells is 18 h, which is longer than that observed in human lymphocytes (4 h), suggesting that the nucleotidase level in CEM-SS cells is lower than in human lymphocytes. A 154-fold accumulation of dGTP in CEM-SS cells in the presence of BCX-1777 and dGuo compared to a 15-fold accumulation of dGTP in human lymphocytes suggests that kinase level is higher in CEM-SS cells compared to human lymphocytes. High kinase and low nucleotidase levels make CEM-SS cells more sensitive to inhibition by BCX-1777 and dGuo than human lymphocytes. Currently, BCX-1777 is in phase I/II clinical trial for the treatment of T cell malignancies.