Randomised clinical trial: anti-viral activity of ANA773, an oral inducer of endogenous interferons acting via TLR7, in chronic HCV.

BACKGROUND: The ANA773 is an oral prodrug of a small-molecule toll-like receptor (TLR)7 agonist. Preclinical and healthy volunteer clinical studies with ANA773 have demonstrated induction of endogenous interferon-α (IFN-α) of multiple subtypes, which supports the potential utility in the treatment of chronic hepatitis C virus (HCV) infection. AIM: To examine safety, tolerability, pharmacodynamics, pharmacokinetics and anti-viral activity of ANA773. METHODS: The ANA773 was investigated in a double-blind, placebo-controlled study in 34 patients chronically infected with HCV of any genotype. Patients were treatment-naïve or had relapsed following previous interferon-based treatment. This dose escalation study was composed of four dose groups (800, 1200, 1600 and 2000mg). In each group, six to eight patients received ANA773 and two received placebo. Patients were dosed with ANA773 every-other-day for either 28 days (800, 1200 or 1600mg) or 10days (2000mg). RESULTS: Mild to moderate adverse events were reported, with an increase in frequency and intensity with increasing dose. No serious AEs were reported and there were no early discontinuations. There were dose-related increases in various markers of IFN-α response. The mean maximum change in serum HCV RNA level from baseline was -0.34, -0.29, -0.40, -0.97 and -1.26log(10) in the placebo, 800, 1200, 1600 and 2000mg cohorts, respectively. At the 2000mg dose, ANA773 significantly (P=0.037) reduced serum HCV RNA levels (range: 0.14 to -3.10log(10) ). CONCLUSION: The ANA773 was generally well tolerated and resulted in a dose-related IFN-dependent response leading to a significant decrease in serum HCV RNA levels in the 2000mg dose group.

The pharmacology of endosomal TLR agonists in viral disease.

The discovery of endosomal TLRs (Toll-like receptors) and their natural ligands has accelerated efforts to exploit them for therapeutic benefit. Importantly, this was preceded by clinical exploration of agents now known to be endosomal TLR agonists. Clinical effects in viral disease have been reported with agonists of TLR3, TLR7, TLR7/8 and TLR9, and the TLR7 agonist imiquimod is marketed for topical use against warts, a papillomavirus disease. The observed pre-clinical and clinical profiles of agonists of each of these TLRs suggest induction of a multifaceted innate immune response, with biomarker signatures indicative of type 1 interferon induction. However, these agents differ in both their pharmaceutical characteristics and the cellular distribution of their target TLRs, suggesting that drugs directed to these targets will display differences in their overall pharmacological profiles.

Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets.

The accurate detection of influenza by clinical symptoms is challenging since multiple pathogenic viruses and bacteria mimic similar symptoms in a patient. With new and more effective influenza therapeutics available, there is a growing need for highly accurate and rapid diagnosis of influenza, particularly when the window of opportunity for proper treatment is measured in hours. A parallel technology, which is also used in the treatment of influenza, was developed for the rapid diagnosis of influenza by exploiting the enzymatic activity of influenza neuraminidase. This technology, which is called Pathozyme, offers the high specificity inherent from the conservation of the neuraminidase active site. The ZstatFlu test uses a small molecule derivative of sialic acid chemically coupled to a reporter group together with simple point-of-care reagents for directly detecting influenza from a patient specimen with high specificity. A second-generation platform technology using this neuraminidase detection system coupled with a more sensitive chemiluminescent reporter has been developed and formatted for reading on high-speed instant film. This modification resulted in a platform technology many-fold more sensitive than the former while maintaining its inherent high specificity. Preliminary data from a prototype tested during the mild 2000-2001 influenza season demonstrated that an optimized chemiluminescent test system could approach the accuracy of 14 day viral culture in a convenient 10-20 min test. This platform technology is currently being explored for the rapid detection of other pathogenic organisms where sensitivity, specificity and speed are essential in a point-of-care setting.

AMP deaminase inhibitors. 3. SAR of 3-(carboxyarylalkyl)coformycin aglycon analogues.

N3-Substituted coformycin aglycon analogues with improved AMP deaminase (AMPDA) inhibitory potency are described. Replacement of the 5-carboxypentyl substituent in the lead AMPDA inhibitor 3-(5-carboxypentyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1, 3]diazepin-8-ol (2) described in the previous article with various carboxyarylalkyl groups resulted in compounds with 10-100-fold improved AMPDA inhibitory potencies. The optimal N3 substituent had m-carboxyphenyl with a two-carbon alkyl tether. For example, 3-[2-(3-carboxy-5-ethylphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4, 5-d][1,3]diazepin-8-ol (43g) inhibited human AMPDA with a K(i) = 0. 06 microM. The compounds within the series also exhibited >1000-fold specificity for AMPDA relative to adenosine deaminase.

AMP deaminase inhibitors. 2. Initial discovery of a non-nucleotide transition-state inhibitor series.

A series of N3-substituted coformycin aglycon analogues are described that inhibit adenosine 5'-monophosphate deaminase (AMPDA) or adenosine deaminase (ADA). The key steps involved in the preparation of these compounds are (1) treating the sodium salt of 6, 7-dihydroimidazo[4,5-d][1,3]diazepin-8(3H)-one (4) with an alkyl bromide or an alkyl mesylate to generate the N3-alkylated compound 5 and (2) reducing 5 with NaBH(4). Selective inhibition of AMPDA was realized when the N3-substituent contained a carboxylic acid moiety. For example, compound 7b which has a hexanoic acid side chain inhibited AMPDA with a K(i) = 4.2 microM and ADA with a K(i) = 280 microM. Substitution of large lipophilic groups alpha to the carboxylate provided a moderate potency increase with maintained selectivity as exemplified by the alpha-benzyl analogue 7j (AMPDA K(i) = 0.41 microM and ADA K(i) > 1000 microM). These compounds, as well as others described in this series of papers, are the first compounds suitable for testing whether selective inhibition of AMPDA can protect tissue from ischemic damage by increasing local adenosine concentrations at the site of injury and/or by minimizing adenylate loss.

Design, synthesis, and structure-activity relationships of the first highly potent, selective, and bioavailable adenosine 5'-monophosphate deaminase inhibitors.

Structure-activity studies have been performed to optimize the potency of this novel series of AMPDA inhibitors. Conformational rigidification of the N-3 sidechain resulted in substantial effect on the potency. Addition of the hydrophobic groups provided further benefit. The most potent compound identified, 4g (Ki = 3 nM), bears little structural resemblance to AMP and exhibits a remarkable improvement (10(3) and 10(5)) in binding affinity relative to the original lead and AMP, respectively. The application of prodrug strategy achieved a large improvement (benzyl ester 5d) in oral bioavailability, resulting in compounds that should be useful in evaluating the role of AMPDA in normo- and pathophysiological states.

Design and synthesis of the first potent, selective, and cell penetrating adenosine 5'-monophosphate deaminase inhibitors.

A major milestone in purine metabolism research has been achieved with the discovery of these potent and selective AMPDA inhibitors. These inhibitors of AMPDA are based on carboxypentyl substitution on N-3 of the coformycin aglycon. They are simpler than coformycin ribose 5'-monophosphate, more stable, selective against other AMP binding enzymes as well as ADA and have good cell penetration and good oral bioavailability. These compounds and their more potent analogs are the first compounds with suitable characteristics to allow a definitive analysis of the role of AMPDA in cellular metabolism and AMPDA as a therapeutic target.

The therapeutic potential of agents acting via purine receptors.

A host of physiological processes associated with the cardiovascular (CV) system, central nervous system (CNS), and a variety of other organ systems and tissues are regulated by agents, primarily adenosine (ado) and adenosine triphosphate (ATP), that act via cell-surface purine receptors. These receptors have therefore been the focus of a variety of programmes directed at the discovery and development of new therapeutic agents, most notably for the treatment of disorders of the CV system. Currently, only a handful of agents, including ado, theophylline, dipyridamole, and ticlopidine, are approved for clinical use. A variety of new agents intended for use in CV disease, disorders of the CNS, such as Parkinson's disease, treatment of pain, inflammatory disorders, and diverse other pathophysiological conditions are in clinical development. Historically, ado receptors have been the primary target. Recent research efforts have begun to examine alternative strategies including agents that modulate endogenous levels of extracellular ado and agents that act via P(2) receptors.

Kinetic scheme for thymidylate synthase from Escherichia coli: determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis.

We have determined kinetic and thermodynamic constants governing binding of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + dUMP --> TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUMP x (6R)-5,10-CH2H4folate --> TS x dTMP x H2folate --> TS x dTMP --> TS as predicted previously by others from steady-state measurements. (2) When substrates are saturating, the overall reaction rate is governed by the slow conversion of enzyme-bound substrates to bound products as demonstrated by (i) large primary and secondary isotope effects on k(cat) and (ii) high rates of product dissociation compared to k(cat). (3) Stopped-flow studies measuring the binding of 10-propargyl-5,8-dideazafolate, an analog of (6R)-5,10-CH2H4folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physical events corresponding to spectral changes which are observed with the wild-type enzyme during initiation of catalysis. A kinetically identifiable reaction step, TS x dUMP x (6R)-5,10-CH2H4folate --> (TS x dUMP x (6R)-5,10-CH2H4folate)*, likely represents reorientation of the C-terminus of the enzyme over the catalytic site. This seals the substrates into a relatively nonaqueous environment in which catalysis can occur. (4) Although TS is a dimer of identical subunits, catalysis is probably confined to only one subunit at a time. (5) The "high-resolution" kinetic scheme described herein provides a framework for the interpretation of the kinetics of catalysis by mutant ecTS chosen to provide insights into the relationship between structure and function.

Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

Critical role of phenylalanine 34 of human dihydrofolate reductase in substrate and inhibitor binding and in catalysis.

Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The variant enzymes are stable and have decreased affinities for methotrexate (by factors of 2700-60000 at pH 7.65) due to a decreased rate of methotrexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether these enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marked inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry and fluorimetry and other methods, it has been shown that, like the wild-type enzyme, the variants have a branched reaction pathway, but in contrast to the wild-type enzyme, the distribution of flux between alternate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociation of tetrahydrofolate from the ternary product complexes of the variant enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the resulting abortive complex. When steady state kinetics for this model are simulated using the experimentally determined rate and dissociation constants for the alanine 34 variant, most steady state experimental results are closely approximated.

Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions.

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.

13C and 15N nuclear magnetic resonance evidence that the active site carboxyl group of dihydrofolate reductase is not involved in the relay of a proton to substrate.

Nuclear magnetic resonance (NMR) spectra for [2-amino,3-15N2]folate and [2-13C]folate complexed with human dihydrofolate reductase, and for complexes of similarly labeled dihydrofolate, show that the N-3 proton of bound folate or dihydrofolate exchanges slowly with solvent and that the bound substrates are in the imino-keto tautomeric form. Previously proposed schemes for substrate protonation that require bound substrate to be in the enolic tautomer are therefore unlikely. The NMR spectra for bound folate are unchanged by raising the pH from 7 to 9.5, whereas those for free folate show marked changes due to ionization for the N-3 proton. The fraction of bound folate with the N-3 proton ionized at pH 9.5 is therefore very small, and the rate constant for the dissociation of the ionized species must be at least 320 times faster than for the protonated species. Comparison of NMR spectra over the pH range 5 to 7 gives no indication of a change in ionization state of the Glu30 carboxyl group over this pH range. This raises doubts about whether the apparent pKa of approximately 6 that describes pH dependence of hydride transfer is due to ionization of this carboxyl group.

Dihydrofolate reductase from the pathogenic fungus Pneumocystis carinii: catalytic properties and interaction with antifolates.

Dihydrofolate reductase (DHFR) from the fungus Pneumocystis carinii (pcDHFR), a target for antifolate inhibitors, has been compared with host enzyme, human DHFR (hDHFR), and with DHFR from Escherichia coli. Among the results of the considerable structural differences between pcDHFR and the other two enzymes is a much higher turnover number (kcat, 136 s-1) for pcDHFR. This is due to rapid hydride transfer from NADPH to dihydrofolate (rate constant 402 s-1), very rapid dissociation of NADP from the product complex (rate constant, k(off), > 1000 s-1), and after NADPH binding, rapid dissociation of tetrahydrofolate (k(off), 216 s-1). Cycling of pcDHFR is almost exclusively by this pathway. The high kcat contributes to a high Km for NADPH (9 microM) and an unusually high Km for dihydrofolate (2.5 microM). Nevertheless, the efficiency of pcDHFR is greater than DHFR from E. coli and about 25% that of hDHFR. Of seven clinically relevant inhibitors investigated, only one (trimethoprim) had a slightly lower Ki for pcDHFR than for hDHFR. The therapeutic value of trimethoprim-sulfa treatment of P. carinii infections indicates that other factors play an important role, but the results are consistent with the frequency of complications due to toxicity of trimethoprim.

Activity of human DNA polymerases alpha and beta with 2-chloro-2'-deoxyadenosine 5'-triphosphate as a substrate and quantitative effects of incorporation on chain extension.

When 2-chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) is incorporated into DNA by human polymerases alpha and beta (Hpol alpha, Hpol beta) the rate of chain extension decreases. In the present study primer extension has been quantitated by estimating the concentration of each successive oligonucleotide product at a series of time points. This has permitted calculation of pseudo-first-order rate constants for successive nucleotide additions to primer. By this method it has been shown that rate constants for CldATP addition are 79-100% of those for dATP in the case of Hpol alpha, and 26-153% with Hpol beta. The concentrations of CldATP for half maximum velocity is 0.6 microM for Hpol alpha, and 6 microM for Hpol beta, each about twice the value for dATP. Thus, CldATP is a good substrate for both enzymes but is more efficiently used by Hpol alpha. Addition of a single analogue residue by Hpol beta to any of seven primers decreases the rate constant for addition of the next nucleotide to 2-7% of that after dAMP addition and further extension is negligible. Consecutive additions of analogue residues by Hpol alpha progressively decrease the rate of subsequent extension, and after five consecutive additions extension virtually terminates. These effects probably make a major contribution to the cytotoxicity of chlorodeoxyadenosine and its therapeutic usefulness as an antileukemic agent.

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.