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Biomedical subjects

F J Dutko

Publications and source records attributed to F J Dutko.

At least 19 recordsLinked to original sources
PubMed1997

Antiviral properties of 3-quinolinecarboxamides: a series of novel non-nucleoside antiherpetic agents.

Novel antiherpetic 3-quinolinecarboxamides were discovered as part of a drug discovery program at Sterling Winthrop Inc. A major goal of this research was to identify novel non-nucleoside agents possessing activity against acyclovir resistant herpes simplex virus. From screening compound libraries in an HSV-2 plaque reduction assay, 1-ethyl-1,4-dihydro-4-oxo-7-(4-pyridinyl)-3-quinolinecarboxamide (1) emerged as an attractive lead structure. By modifying the quinoline ring at the 1-, 2-, 3-, 4-, and 7-positions, analogues were identified that have up to 5-fold increased in vitro potency relative to acyclovir. In a single dose mouse model of infection the 1-(4-FC6H4) analogue 17, one of the most potent derivatives in vitro, displayed comparable oral antiherpetic efficacy to acyclovir at 1/16 the dose; in a multiple dose regimen, however, it was 2-fold less potent. Mechanism of action studies indicate that these new compounds interact with a different, as yet undefined, molecular target than acyclovir.

Acyclovir↗
PubMed1995

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND: Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS: The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS: The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

Amino Acid Sequence↗
PubMed1995

Structures of four methyltetrazole-containing antiviral compounds in human rhinovirus serotype 14.

Four novel antiviral WIN compounds, that contain a methyl tetrazole ring as well as isoxazole, pyridazine or acetylfuran rings, have had their structures determined in human rhinovirus serotype 14 at 2.9 A resolution. These compounds bind in the VP1 hydrophobic pocket, but are shifted significantly towards the pocket pore when compared to previously examined WIN compounds. A putative water network at the pocket pore is positioned to hydrogen bond with these four WIN compounds, and this network can account for potency differences seen in structurally similar WIN compounds.

Journal Article↗
PubMed1995

Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism.

Several modifications of the oxazoline ring of WIN 54954, a broad spectrum antipicornavirus compound, have been prepared in order to address the acid lability and metabolic instability of this compound. We have previously shown that the oxadiazole analogue 3 displayed comparable activity against a variety of rhinoviruses and appeared to be stable to acid. A monkey liver microsomal assay was developed to examine the metabolic stability in vitro of both compounds, and it was determined that WIN 54954 displayed 18 metabolic products while 3 was converted to 8 products. Two major products of 3 were determined by LC-MS/MS to be monohydroxylated at each of the terminal methyl groups. Replacement of the methyl on the isoxazole ring with a trifluoromethyl group, while preventing hydroxylation at this position, did not reduce the sensitivity of the molecule to microsomal metabolism at other sites. However, the (trifluoromethyl)oxadiazole 9 not only prevented hydroxylation at this position but also provided protection at the isoxazole end of the molecule, resulting in only two minor products to the extent of 4%. The major product was identified as the monohydroxylated compound 23. The global metabolic protective effect of trifluoromethyl group on the oxadiazole ring was further demonstrated by examining a variety of analogues including heterocyclic replacements of the isoxazole ring. In each case, the trifluoromethyl analogue displayed a protective effect when compared to the corresponding methyl analogue.

Animals↗
PubMed1994

An evaluation of the antirhinoviral activity of acetylfuran replacements for 3-methylisoxazoles. Are 2-acetylfurans bioisosteres for 3-methylisoxazoles?

As a probe of the 3-methylisoxazole portion of our broad-spectrum antipicornaviral series, a panel of 2-acetylfuran analogues was prepared as replacements for the 3-methylisoxazole ring. Comparison of the two series showed remarkable similarity in potency, spectrum of activity, logP, and electrostatic parameters. X-ray studies of 21b bound to human rhinovirus-14 showed that the 2-acetyl group adopted a syn conformation and the carbonyl oxygen acts as a hydrogen bond acceptor with ASN219 in much the same way as the nitrogen of the isoxazole. The importance of the syn conformation and the hydrogen-bonding capability was confirmed by the reduced antiviral activity of the 2-methylfuran and 2-formylfuran analogues. From the results of this study, it is apparent that the syn-2-acetylfuran ring is acting as a bioisostere for the 3-methylisoxazole.

Antiviral Agents↗
PubMed1994

Oxadiazoles as ester bioisosteric replacements in compounds related to disoxaril. Antirhinovirus activity.

A series of 1,2,4-oxadiazoles has been prepared as ester bioisosteres and tested against 15 human rhinovirus serotypes, and the MIC80, the concentration which inhibits 80% or 12 of the serotypes tested, was determined. Homologation of the alkyl group attached to the oxadiazole ring resulted in a reduction in activity with increased chain length. Introduction of hydrophilic groups in this position rendered the compounds inactive. Increasing the length of the side chain attached to the isoxazole ring resulted in an increase in activity. Replacement of the methyl with alkoxyalkyl substituents retained activity; however, introduction of a hydroxyl group on to the side chain reduced activity. Compound 8a, where both the isoxazole and oxadiazole rings were substituted with methyl groups, was one of the most active compounds in the series. A comparison was made between 8a and the two isomeric oxadiazoles 41 and 46, and an attempt was made to explain the difference in activity by examining electrostatic potential maps and by an energy profiling study. No conclusive results were obtained from these studies.

Antiviral Agents↗
PubMed1994

Human rhinovirus 14 complexed with fragments of active antiviral compounds.

Crystallographic studies of human rhinovirus 14 (HRV14) crystals soaked with fragments of antiviral WIN compounds, at high concentrations (82-200 micrograms/ml), show the compounds bind into the hydrophobic beta-barrel (WIN pocket) of VP1. Two of these short compounds (5-[3,5-dimethyl-4-hydroxyphenyl]-2-methyltetrazole and phenol oxazoline) cause conformational changes in the virus similar to the active, longer WIN compounds. In addition, thermostabilization studies suggest these short WIN compounds provide some stability to the HRV14 capsid. We conclude that the short compounds appear to mimic the cellular cofactors observed in the hydrophobic pocket of VP1 for some picornaviruses. Both cofactors and short WIN compounds bind into the pocket, cause conformational changes in VP1, and provide a small degree of virion stabilization but are unlikely to inhibit attachment. Three specific binding sites for dimethyl sulfoxide (DMSO), used as solvent, were also identified. One of the DMSO molecules binds into the drug binding pocket near the pocket opening, while the other two bind in the canyon near the VP1 protomer-protomer interface.

Antiviral Agents↗
PubMed1993

Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme.

Bacterial beta-galactosidase is one of several reporter enzymes used in studying the transcriptional activity of eukaryotic promoters. Although it is one of the easiest and least expensive enzymes to assay, its use has been limited because of its low sensitivity, which is due in part to endogenous levels of beta-galactosidase in many eukaryotic cells. In this study, we compared the pH and salt requirements, as well as the heat stability, of bacterial and eukaryotic beta-galactosidase in order to identify conditions which would inhibit the beta-galactosidase enzyme endogenous to eukaryotic cells without adversely affecting the activity of either purified bacterial beta-galactosidase or reporter beta-galactosidase produced after transfection of expression vectors into eukaryotic cells. Heat treatment at 50 degrees C for 1 h inactivated the beta-galactosidase activity endogenous to several eukaryotic cell lines by as much as 40-fold without adversely affecting the activity of bacterial beta-galactosidase. This treatment increased the sensitivity of this reporter enzyme and allowed the development of a rapid and quantifiable screening assay for HIV-1 tat inhibitors.

Animals↗
PubMed1993

The structure of human rhinovirus 16.

BACKGROUND: Rhinoviruses and the homologous polioviruses have hydrophobic pockets below their receptor-binding sites, which often contain unidentified electron density ('pocket factors'). Certain antiviral compounds also bind in the pocket, displacing the pocket factor and inhibiting uncoating. However, human rhinovirus (HRV)14, which belongs to the major group of rhinoviruses that use intercellular adhesion molecule-1 (ICAM-1) as a receptor, has an empty pocket. When antiviral compounds bind into the empty pocket of HRV14, the roof of the pocket, which is also the floor of the receptor binding site (the canyon), is deformed, preventing receptor attachment. The role of the pocket in viral infectivity is not known. RESULTS: We have determined the structure of HRV16, another major receptor group rhinovirus serotype, to atomic resolution. Unlike HRV14, the pockets contain electron density resembling a fatty acid, eight or more carbon atoms long. Binding of the antiviral compound WIN 56291 does not cause deformation of the pocket, although it does prevent receptor attachment. CONCLUSIONS: We conjecture that the binding of the receptor to HRV16 can occur only when the pocket is temporarily empty, when it is possible for the canyon floor to be deformed downwards into the pocket. We further propose that the role of the pocket factor is to stabilize virus in transit from one host cell to the next, and that binding of ICAM-1 traps the pocket in the empty state, destabilizing the virus as required for uncoating.

Amino Acid Sequence↗
PubMed1993

Efficacy of oral WIN 54954 for prophylaxis of experimental rhinovirus infection.

The efficacy of oral WIN 54954 for the prevention of rhinovirus infection and illness was tested in two randomized, double-blinded, placebo-controlled volunteer challenge studies. Volunteers were inoculated with rhinovirus type 39 (MIC of WIN 54954, 0.17 microgram/ml) or rhinovirus type 23 (MIC, 0.016 microgram/ml). The volunteers received two doses of drug (600 mg per dose) or placebo on the first day; this was followed by three doses on each of the subsequent 5 days. All volunteers were challenged with virus after the third dose of study drug. No significant antiviral or clinical effect was detected in either study. Pharmacokinetic studies revealed that on the last day of drug administration, 38 of 39 (97%) volunteers had trough levels of WIN 54954 in plasma greater than the MIC for the respective virus. Nasal wash specimens collected on the same day revealed a detectable level in only 6 of 24 (25%) volunteers at the peak (range, 6 to 24 ng/ml) and in only 2 of 14 (14%) volunteers at the trough (range, 6 and 7 ng/ml). These results suggest that the lack of efficacy of WIN 54954 against rhinovirus may be related to an inability to deliver sufficient drug to the site of viral infection.

Adult↗
PubMed1992

Conformationally restricted analogues of disoxaril: a comparison of the activity against human rhinovirus types 14 and 1A.

A series of conformationally restricted analogs of disoxaril has been synthesized and evaluated against human rhinovirus types (HRV) 14 and 1A. The sensitivity of these serotypes to this series varied and was dependent upon the length of the molecule as well as upon the flexibility of the aliphatic chain. Minimum energy conformations of these compounds were overlaid with the X-ray structure of a closely related analog 9 bound to the capsid protein of both HRV-14 and -1A and then modeled in the compound-binding site of both serotypes. A comparative sweep volume of these compounds about the isoxazole ring revealed an inaccessible region of space for the cis-olefin 8b, which is not the case for either the trans-olefin 8a or the acetylene 5. This region may be important to the binding of the compounds to the HRV-14 site particularly during entry into the pocket.

Antiviral Agents↗
PubMed1992

CoMFA analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14.

A CoMFA analysis of eight compounds related to disoxaril whose X-ray structures bound to HRV-14 had been determined resulted in a strong positive correlation of activity with steric effects of the compounds, particularly toward the pore end of the compound binding site, and no correlation with electrostatic effects. These results confirm what had been previously found, that the activity of these compounds was highly dependent upon their hydrophobic nature as expressed by log p. The CoMFA study also confirmed the results from the comparison of a series of active and inactive compounds using volume maps which showed that bulk at the pore end of the molecule was conducive to high levels of antiviral activity while excessive bulk around the ring led to poor activity.

Antiviral Agents↗
PubMed1992

Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound.

Self-emulsifying drug delivery systems (SEDDSs) represent a possible alternative to traditional oral formulations of lipophilic compounds. In the present study, a lipophilic compound, WIN 54954, was formulated in a medium chain triglyceride oil/nonionic surfactant mixture which exhibited self-emulsification under conditions of gentle agitation in an aqueous medium. The efficiency of emulsification was studied using a laser diffraction sizer to determine particle size distributions of the resultant emulsions. An optimized formulation which consisted of 25% (w/w) surfactant, 40% (w/w) oil, and 35% (w/w) WIN 54954 emulsified rapidly with gentle agitation in 0.1 N HCl (37 degrees C), producing dispersions with mean droplet diameters of less than 3 microns. The self-emulsifying preparation was compared to a polyethylene glycol 600 (PEG 600) solution formulation by administering each as prefilled soft gelatin capsules to fasted beagle dogs in a parallel crossover study. Pharmacokinetic parameters were determined and the absolute bioavailability of the drug was calculated by comparison to an i.v. injection. The SEDDS improved the reproducibility of the plasma profile in terms of the maximum plasma concentration (Cmax) and the time to reach the maximum concentration (tmax). There was no significant difference in the absolute bioavailability of WIN 54954 from either the SEDDS or the PEG formulations.

Administration, Oral↗
PubMed1991

Binding affinities of structurally related human rhinovirus capsid-binding compounds are related to their activities against human rhinovirus type 14.

The binding affinities (Kds) and the rates of association and dissociation of members of a chemical class of antiviral compounds at their active sites in human rhinovirus type 14 (HRV-14) were determined. On the basis of analysis by LIGAND, a nonlinear curve-fitting program, of saturation binding experiments with HRV-14, the Kds for Win 52084, Win 56590, disoxaril (Win 51711), and Win 54954 were found to be 0.02, 0.02, 0.08, and 0.22 microM, respectively. The independently determined kinetic rates of association and dissociation resulted in calculated Kd values which were in agreement with the Kd values determined in saturation binding experiments. Scatchard plots of each of four compounds for the binding data indicated that approximately 40 to 60 molecules were bound per HRV-14 virion. Hill plots showed no evidence of cooperativity in binding. Furthermore, the antiviral activities (MICs in plaque reduction assays with HRV-14) for this limited series of compounds (n = 4) correlated well (r = 0.997) with the observed Kds. Likewise, the absence of detectable binding of Win 54954 to the drug-resistant mutant HRV-14 (Leu-1188) corresponded to a lack of antiviral activity. The positive relationship between the antiviral activities and the Kds that were determined may have implications for the molecular design of capsid-binding antirhinovirus drugs.

Antiviral Agents↗
PubMed1990

A model for compounds active against human rhinovirus-14 based on X-ray crystallography data.

A number of (oxazolinylphenyl)isoxazoles have been synthesized and tested against human rhinovirus-14 (HRV-14). Several of the more active compounds have been examined by X-ray crystallography and their orientation in the compound binding site on the capsid protein of HRV-14 has been determined. Based on the minimum inhibitory concentration against HRV-14 and the X-ray conformation of the compounds, a model has been developed which distinguishes between the space-filling properties of the active and inactive compounds in this series. The model was generated by overlaying composite structures and comparing the van der Waals generated volume maps. The results of this study indicate that inactive compounds display areas of excessive bulk particularly around the phenyl ring, while the active compounds occupy space below the pore area of the compound binding site.

Antiviral Agents↗
PubMed1989

Crystal structure of human rhinovirus serotype 1A (HRV1A).

The structure of human rhinovirus 1A (HRV1A) has been determined to 3.2 A resolution using phase refinement and extension by symmetry averaging starting with phases at 5 A resolution calculated from the known human rhinovirus 14 (HRV14) structure. The polypeptide backbone structures of HRV1A and HRV14 are similar, but the exposed surfaces are rather different. Differential charge distribution of amino acid residues in the "canyon", the putative receptor binding site, provides a possible explanation for the difference in minor versus major receptor group specificities, represented by HRV1A and HRV14, respectively. The hydrophobic pocket in VP1, into which antiviral compounds bind, is in an "open" conformation similar to that observed in drug-bound HRV14. Drug binding in HRV1A does not induce extensive conformational changes, in contrast to the case of HRV14.

Amino Acid Sequence↗