Characterization of the binding affinities of peramivir and oseltamivir carboxylate to the neuraminidase enzyme.

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme-inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.

A single intramuscular injection of neuraminidase inhibitor peramivir demonstrates antiviral activity against novel pandemic A/California/04/2009 (H1N1) influenza virus infection in mice.

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes--a novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies.

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC(50) of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 microM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC(50)s of 0.159, 0.26 and 0.73 microM, respectively. The IC(50) for dGuo in the presence of 1 microM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 microM. dGTP in human lymphocytes is elevated and a 3-5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 microM (predose levels<0.004 microM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

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.

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.

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.

Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors.

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

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.