Synthesis and evaluation against hepatitis C virus of 7-deaza analogues of 2'-C-methyl-6-O-methyl guanosine nucleoside and L-Alanine ester phosphoramidates.

7-Deazapurines are known to possess broad antiviral activity, however the 2'-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2'-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2'-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2'-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2'-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue.

INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties.

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus.

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.

New malaria chemotherapy developed by utilization of a unique parasite transport system.

During its development in the host red cell, the human malarial parasite causes profound alteration in the permeability of the host cell membrane. These membrane transport systems(s) play a role in the development of the intra-erythrocytic parasite in its need to take up solutes and nutrients from the extracellular medium and the disposal of metabolic wastes. Importantly, the properties of these parasite induced transport systems are significantly different from those in normal human cells. Hence, such systems are of considerable interest for their potential use in anti-malarial chemotherapy, both by (i). inhibiting the transport and hence depriving the parasite of nutrients essential for its development, or (ii). by designing cytotoxic drugs which selectively enter the parasite through these induced transporter routes and hence cannot enter normal mammalian cells. Since our discovery that optical isomers of nucleosides (such as L- adenosine or L- thymidine) were selectively transported into malaria infected cells through the induced transporter, L-nucleoside drug "carriers" were actively synthesized as potentially new therapeutic agents. The compounds are dinucleoside phosphate dimers, where each "carrier" (a L-nucleoside) has been conjugated to known anti-malarial agents, such as 5'-fluro-uridine through the 3' and 5'-OH and a phosphate group. A very large series of these drugs have been synthesized with varying conjugations. The dimers are extremely toxic against malaria and experimental evidence has confirmed that they are incapable of entering normal mammalian cells. This review discusses their mechanism of action and potential as new anti-malarial chemotherapy as well as the role played by the membrane transport system of malaria infected cells as a target for malaria chemotherapy.

Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-HCV agents.

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 µM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties.

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

A panel of monoclonal antibodies recognizing the Staphylococcus epidermidis fibrinogen-binding MSCRAMM SdrG.

Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall-anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.

Monoclonal antibodies recognizing the Enterococcus faecalis collagen-binding MSCRAMM Ace: conditional expression and binding analysis.

Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.

Human immunoglobulin G recognizing fibrinogen-binding surface proteins is protective against both Staphylococcus aureus and Staphylococcus epidermidis infections in vivo.

A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations.

Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus.

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.

SdrX, a serine-aspartate repeat protein expressed by Staphylococcus capitis with collagen VI binding activity.

Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis.

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A.

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.