Poly-L-proline type II peptide mimics as probes of the active site occupancy requirements of cGMP-dependent protein kinase.

Based on the X-ray crystal structure of cAMP-dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X-ray crystal structure of cyclin-dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly-L-proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP-dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P-1, P-2, and P-3 residues of substrate peptides bind in the PPII conformation (phi approximately -75 degrees, psi approximately 145 degrees). In addition, the inhibition data also suggest that the P-1, P-2, and P-3 residues in substrate peptides bind with a gauche(-) chi1 angle.

Poly-L-proline type II peptide mimics based on the 3-azabicyclo[3.1.0]hexane system.

This paper describes conformational studies of proline-templated amino acids (PTAAs) based on the 3-azabicyclo[3.1.0]hexane system as well as conformational studies on short peptides composed of these PTAAs. NOE data, coupling constants, and molecular modeling are consistent with a flattened boat conformation for monomeric and oligomeric residues based on this bicyclic system. NMR studies on dimeric and trimeric oligomers are consistent with a populated poly-L-proline type II conformation in CDCl3 and D2O. Solution studies and molecular modeling predicts phi approximately -70 degrees, psi approximately 131 degrees, chi 1 approximately -57 degrees, and chi 2 approximately -158 degrees for oligomeric residues.

Solid-phase guanidinylation as a diversification strategy of poly-L-proline type II peptide mimic scaffolds.

[reaction: see text] Solid-phase guanidinylation of proline-templated amino acids is studied as a diversification strategy of poly-L-proline type II scaffolds.

Structure-activity relationship for DNA topoisomerase II-induced DNA cleavage by azatoxin analogues.

Eighteen analogues of the nonintercalative DNA topoisomerase II (topo II)-active epipodophyllotoxin-ellipticine hybrid, azatoxin, were synthesized and evaluated for their ability to induce topo II-mediated DNA strand breaks in vitro. In general, the SAR profile of the azatoxins showed more homology with that of the epipodophyllotoxins than with the ellipticines. Of the compounds studied, only fluoro substitution at the 8-, 9, and 10-positions of azatoxins enhanced activity, with 9-fluoroazatoxin being the most active compound in this series.

Inhibition of DNA topoisomerase II by azaelliptitoxins functionalized in the variable substituent domain.

A series of novel C11-substituted derivatives of azaelliptitoxin (azatoxin) have been synthesized and tested for their inhibitory activity against human DNA topoisomerase II. Incorporation of a C11 polyamine or amine resulted in an increase in the intercalation properties of the drug and a decrease of topoisomerase II activity. The structure-activity relationship (SAR) profile of the nonintercalating C11 anilino azatoxin class follows the SAR of the (anilino)acridine family. 11-(4-Cyanoanilino)azatoxin (14) was found to be the most active analog in this series, exhibiting approximately 10-fold higher activity than azatoxin 12 and etoposide.

Azatoxin derivatives with potent and selective action on topoisomerase II.

Azatoxin was rationally designed as a DNA topoisomerase II (top2) inhibitor [Leteurtre et al., Cancer Res 52: 4478-4483, 1992] and was also found to inhibit tubulin polymerization. Its cytotoxicity is due to action on tubulin at lower concentrations and on top2 at higher concentrations. At intermediate concentrations, the combination of the two mechanisms appears antagonistic [Solary et al., Biochem Pharmacol 45: 2449-2456, 1993]. The aim of this study was to design azatoxin derivatives that would act only on tubulin or on top2. Selective targeting of top2 or tubulin was tested using top2-mediated DNA cleavage assays, and tubulin polymerization and tubulin proteolysis assays, as well as COMPARE analyses of cytotoxicity assays in the National Cancer Institute in vitro Drug Screening Program. Selective inhibitors of top2 and tubulin polymerization have been obtained. Top2 inhibition, abolished by methylation at position 4', was enhanced by the addition of a bulky group at position 11. Bulky substitution at position 11 determined different patterns of top2 cleavage sites and suppressed the action on tubulin. Selective inhibition of tubulin was obtained with 4'-methylazatoxin that was found to bind to the colchicine site. These results are consistent with those obtained in the podophyllotoxin family to which azatoxin is structurally related. Some azatoxin derivatives are under consideration for further preclinical development.

Rational design and molecular effects of a new topoisomerase II inhibitor, azatoxin.

Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydr oxyphenyl) oxazolo (3',4':1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.

Ecologic studies of Venezuelan encephalitis virus in Peru during 1970-1971.

Venezuelan encephalitis (VE) virus has intermittently produced epidemics and equine epizootics on the dry Pacific coastal plain of Peru since at least the 1930's. However, evidence that the virus exists in the Amazon region of Peru to the east of the Andes mountains was not obtained until antibodies were found in human sera collected in 1965, and 10 strains of the virus were isolated in a forest near the city of Iquitos, Peru during February and March 1971. Eight strains came from mosquitoes and two from dead sentinel hamsters. Three hamsters exposed in forests near Iquitos developed VE virus antibodies suggesting that hamster-benign strains also exist there. Antibody tests of equine sera revealed no evidence that VE virus was actively cycling during the late 1950's or 1960's in southern coastal Peru, where equine epizootics had occurred in the 1930's and 1940's. In northern coastal Peru bordering Ecuador, antibodies were present in equine sera, presumably residual from the 1969 outbreak caused by subtype I virus, since neutralizing antibody titers were higher to subtype I virus than to subtypes III or IV. No VE virus was detected in this northern region during the dry season of 1970 by use of sentinel hamsters. The possibility is considered that VE epidemics and equine epizootics on the Pacific coast of Peru are caused by movements of virus in infected vertebrates traversing Andean passes or in infected vertebrates or mosquitoes carried in airplanes from the Amazon region.

The first isolations of eastern encephalitis, group C, and Guama group arboviruses from the Peruvian Amazon region of western South America.

Two strains of eastern encephalitis (EE) virus were isolated in the Amazon region of Peru near Pucallpa, Loreto Department, using sentinel hamsters. EE virus antibodies were found in healthy horses at both Pucallpa and Iquitos in the same Department. Fourteen group C and four Guama group arboviruses were recovered from sentenel hamsters and mosquitoes near Iquitos. The group C agents were Caraparu-Ossa, Marituba, and Oriboca-Itaqui viruses, and the Guama group agents were Bimiti virus. Besides providing a detailed account of these investigations, this article includes a current list of known arboviruses of the American tropics that can be detected with sentinel hamsters.

The prevalence of hepatitis B antigen among hepatitis patients and residents of Peru.

Single radial immune diffusion and counterelectrophoresis tests were used to examine 2,593 serum speciments from apparently healthy men, women, and children of Peru for the presence of hepatitis B antigen (Australia antigen). The object was to estimate the prevalence of the antigen in two contrasting geographic regions and to investigate the relationship between presence of the antigen and jungle hepatitis in eastern Peru. In connection with the latter goal, both single and serial serum samples collected from hepatitis cases during an epidemic in the northeast were also examined. Of the 2,593 apparently healthy subjects, the tests showed 1.4 per cent were carrying the antigen. However, when the data were broken down by geographic region it was found that 1.8 per cent of the subjects from eastern Peru were carriers, as compared to only 0.5 per cent of those from the northern coast. Moreover, incidences as high as 5 and 6.4 per cent were found in selected eastern areas, and a peak figure of 14.3 per cent was found in sera from children living in some of these areas. Comparison of the proportions of male and female sera positive for the antigen indicated that the proportion of males with HB Ag was nearly twice as high. However, sera collected from Indians of 20 different eastern tribes and from mestizos in the eastern region showed roughly the same proportion of samples positive for HB Ag in each ethnic group. The study also showed a close correlation between presence of the antigen and hepatitis infection during a 1972-1973 epidemic in eastern Peru. Testing of sera taken from hepatitis patients at that time showed many patients to be carrying HB Ag, specially in cases where serial blood samples were available. In all, positive test results were obtained for 81.2 per cent of the patients from whom two or more samples had been obtained.

Arbovirus neutralization tests with Peruvian sera in Vero cell cultures.

Selected human sera from Peru, previously examined by the haemagglutination-inhibition (HI) test with a number of arboviruses, were reexamined by neutralization tests carried out in Vero cell cultures. Results confirmed and extended the HI findings, indicating that the antibodies detected were evoked by Eastern equine encephalitis, Mayaro, Venezuelan equine encephalitis, Ilheus, St Louis encephalitis, yellow fever, Caraparu, and Guaroa viruses.

WHO collaborative study on the sero-epidemiology of rubella in Caribbean and Middle and South American populations in 1968.

Knowledge of the rubella antibody profiles of female populations of various ages and in various geographical areas is essential for an intelligent and effective administration of rubella vaccine. The investigation reported was undertaken to extend a previous WHO collaborative study to include additional areas of the Americas. As in the other mainland areas included in the earlier study, the presence of rubella haemagglutination-inhibiting antibody was found to be a likely event in over 80% of the females of child-bearing age in Argentina, Brazil, Chile, urban Peru (Lima) and Uruguay. Antibody rates were significantly lower in Jamaica, Panama, rural Peru and Trinidad. These data confirm and extend earlier findings of low levels of rubella immunity in certain island or isolated populations.