Biomedical subjects
Publications and source records attributed to S Massa.
Thiomorpholine analogues of U-100480 with the biphenylmethyl group replacing the acetamidomethyloxazolidinone moiety have been synthesized and tested as antimycobacterial agents together with various related derivatives. Some biphenyl derivatives were endowed with high activity against Mycobacterium tuberculosis and other non-tuberculous mycobacteria.
One-day old chickens were treated with a faecal suspension and faecal samples were cultivated either in non-selective broth (Viande Levure broth) or in broths selective for Lactobacillus spp., Bacteroides spp., faecal enterococci and coliforms. The chickens were infected 48 h later with 10(4) colony-forming units of nalidixic-acid resistant Salmonella kedougou and examined seven days later. Faecal suspensions and faecal samples cultivated in non-selective broth exerted a protection against Salmonella infection; no protection was obtained with faecal samples cultivated in selective broths, administered either alone or in combination.
The intensity of nonspecific immune reaction and the host resistance to facultative intracellular pathogens are found to be associated in lines of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity. AIRmax are more resistant than AIRmin mice to Salmonella typhimurium and Listeria monocytogenes infection, the differences between lines in LD50 being > 1000 and 100 times, respectively. This difference was shown to be related to the initial bacterial containment at the infectious focus, and to the control of bacterial multiplication in the spleen during the 1st week after s. c. inoculation of the bacteria. Specific immune responses were not deeply affected by the selective process: antibody production and delayed-type hypersensitivity were both of similar intensity in AIRmax and AIRmin mice. The differential susceptibility to infection seems independent of the Nramp-1 locus polymorphism; therefore, these two lines represent a powerful model for investigating the role of other genetic loci regulating the nonspecific immunity effectors in the course of infectious diseases.
Two distinct bidirectional selective breedings for quantitative traits were initiated from identical genetically heterogeneous mouse populations. The resulting lines are characterized by maximal or minimal acute inflammatory responsiveness (AIR): AIRmax and AIRmin lines, respectively, and by resistance or susceptibility to chemical skin tumorigenesis: Car-R and Car-S lines, respectively. The AIR response to s.c. injection of polyacrylamide microbeads, measured by cell content in the local exudate, was 10 times higher in AIRmax than in AIRmin mice. The response to selection was asymmetrical: the realized heritability was 0.26 in AIRmax and 0.008 in AIRmin, and resulted from the additive effect of 7-11 quantitative trait loci (QTL). Low responsiveness was globally dominant in F1 and 48% of F2 segregant variance was found to be due to genetic factors. These findings are the first demonstration of innate regulation of AIR by germ line genes. Susceptibility to skin tumorigenesis induced by a two-stage initiation (DMBA)-promotion (TPA) protocol was lower in AIRmax mice than in AIRmin mice, a 6-fold difference in tumor induction rate. Intense AIR was found to be associated with resistance, and low AIR with susceptibility to tumorigenesis, in F2 segregants chosen for extreme AIR phenotypes. At least some of the AIR QTLs therefore contain genes controlling tumorigenesis. Tumor phenotypes differed more in Car-R and Car-S than in AIRmax and AIRmin lines, indicating that QTLs unrelated to AIR, contribute to the host response to tumorigenesis. The extreme phenotypes/genotypes of the four selected lines and the known genetic constitution of their foundation population, offer new possibilities to discriminate the genes/mechanisms controlling two important traits: AIR and response to chemical tumorigenesis. Collaborative projects will be favorably considered. The description of tumor resistance genes in AIRmax and Car-R mice may be helpful for epidemiology and therapy of human cancer.
We synthesized and tested as novel inhibitors of human immunodeficiency virus type 1 (HIV-1) bi- and tricyclic thiadiazine ring homologues of 7-chloro-2-ethyl-2H-1,2,4-benzothiadiazin-3-(4H)-one 1,1-dioxide, which is a compound endowed with anti-HIV-1 activity at low micromolar concentrations. Benzothiadiazepine derivatives were obtained by alkylation of 8-chloro-2,3-dihydro-3-methyl-1,2,5-benzothiadiazepin-4(5H)-one 1,1-dioxide, which was obtained by intramolecular cyclization of 2-(2-amino-5-chloro-benzenesulphonamido) propanoic acid. Pyrrolobenzothiadiazepines were synthesized from N-substituted 5-chloro-2-(1H-pyrrol-1-yl)benzene-sulphonamides by treating with triphosgene. N6-substituted pyrrolo[2,1-d][1,2,5]benzothiazepin-7(6H)-one 5,5-dioxides were active, though not very potent. Both a chlorine atom and an unsaturated alkyl chain were found to be determinants of anti-HIV-1 activity. The highest potency was shown by a derivative with a TIBO-related 3,3-dimethylallyl chain. 2,3-Dihydro-1,2,5-benzothiadiazepin-4(5H)-one 1,1-dioxides were scarcely active in HIV-1-infected MT-4 cell assays; however, the introduction of the dimethylallyl chain into 7-chloro-1,2,5-benzothiadiazepine moiety led to a bicyclic derivative which was more potent and less cytotoxic than the tricyclic pyrrole-containing counterpart.
In the presence of sodium hydride, reaction of aryl-disulphides with ethyl esters of indole-2-carboxylic acids furnished ethyl 3-arylthioindole-2-carboxylates, which were cyclized intramolecularly to afford 5H-indolo[3,2-b][1,5]benzothiazepin-6(7H)-ones or hydrolysed in alkaline medium to give 3-arylthioindole-2-carboxylic acids. These acids, also obtained by the action of aryldisulphides on indole-2-carboxylic acids, afforded tetracyclic 5H-indolo [3,2-b][1,5]benzothiazepin-6(7H)-ones upon treatment with EDCI-DMAP. Transformation of cyclic sulphides into the required sulphones was achieved by treatment with hydrogen peroxide or with m-chloroperbenzoic acid. The title derivatives are conformationally constrained analogues of the potent human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor 3-benzene-sulphonyl-5-chloroindole-2-carboxamide (L-737, 126). Although the indolobenzothiazepine derivatives, as well as the indolyl aryl sulphones used for their synthesis, were endowed with anti-HIV-1 activities in the submicromolar and micromolar range, none of them proved more potent than L-737,126.
Novel compounds related to 2-(cyclohexylthio)-3,4-dihydro-5-methyl-6-(3-methylbenzyl)-4-ox opyrimidine (3c, MC 639) have been synthesized and tested as inhibitors of human immunodeficiency virus type-1 (HIV-1). Reaction of thiourea with ethyl arylmethylacetoacetates furnished 5-alkyl-6-(arylmethyl)-3,4-dihydro-2-mercapto-4-oxopyrimidines which were then alkylated at the sulfur atom to afford the required 2-alkylthio or 2-cycloalkylthio derivatives (S-DABOs). Chemical modifications at N-3, C-4, and C-6 of the pyrimidine ring were attempted with the aim of improving antiretroviral activity. In particular, replacement of the benzyl group with the 1-naphthylmethyl moiety enhanced the activity of S-DABOs, whereas N-3 alkylation and C=O transformation into C=S at position 4 of the pyrimidine ring led to compounds devoid of anti-HIV-1 activity. Lower activity was generally observed when 1-naphthylmethyl was replaced by the isomeric 2-naphthylmethyl moiety. The most active compounds showed activity in the low micromolar range with EC50 values comparable to that of nevirapine.
Many and different probiotic pharmaceutical products are presently commercialised in the world. On this regard, a microbiological investigation was carried out to screen the microorganisms incorporated into these products, commonly used for human health. After determination of the cell number and viability of bacteria, several experiments were performed in vitro in order to characterise the microorganisms and to evaluate their probiotic value. Among all the strains identified, best results were obtained with Lactobacillus rhamnosus, Enterococcus faecium and Saccharomyces cerevisiae as far as regards growth rates, pH and bile salts tolerance. Moreover, the identification profiles of microorganisms showed a better reliability for the products containing a single species whereas the ones composed of different strains were usually not satisfactory. In some cases, the presence of Lactobacillus and Saccharomyces species was in disagreement with the claimed composition of the product and some species of lactobacilli, bifidobacteria and streptococci were found not viable. In defined mixed cultures experiments, the antagonism of Lactobacillus acidophilus and Enterococcus faecium versus Yersinia enterocolitica was demonstrated and explained as acid and/or antimicrobials production.
Cow's milk was inoculated with ca 10(3) and 10(7) cfu ml-1 Escherichia coli O157:H7. After fermentation at 42 degrees C for 0-5 h, the yoghurt was stored at 4 degrees C. Two kinds of yoghurt were used: traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum. After 7 d E. coli O157:H7 decreased from 3.52 to 2.72 log10 cfu ml-1 and from 7.08 to 5.32 log10 cfu ml-1 in TY, and from 3.49 to 2.73 log10 cfu ml-1 and from 7.38 to 5.41 log10 cfu ml-1 in BY. The pH values of yoghurt dropped from 6.6 to 4.5 and 4.4 in TY (for low and high pathogen inocula, respectively), and from 6.6 to 4.6 and 4.5 in BY (for low and high pathogen inocula, respectively).
Following the discovery of anti-HIV properties of suramin great efforts were devoted to design novel NNRT agents with the aims to find novel drugs for the clinical therapeutic management of AIDS. Sulfone and sulfonamide derivatives were studied by NCI at Bethesda as potential anti-HIV-1 agents and nitrophenyl phenyl sulfone (NPPS) was selected as lead compound for further investigations. At the same time Merck Laboratories discovered L-737,126, a potent indolyl aryl sulfone with inhibitory activity against reverse transcriptase. These studies stimulated novel search in the sulfone series and both diarylsulfones and cyclic sulfone derivatives were investigated. Our decennial interest in chemotherapeutic agents containing a pyrrole ring pulsed us to synthesize and test as anti-HIV-1 agents a number of pyrryl aryl sulfones (PASs), pyrrolobenzothiadiazepine (PBTDs) and pyrrolobenzothiazepine related sulfones. The new sulfone derivatives inhibit selectively HIV-1 and were inactive against HIV-2. Most of them were as active as, if not more active than, nevirapine.
The B cell-specific activity of immunoglobulin (Ig) gene promoters is to a large extent mediated by the conserved octamer motif ATTTGCAT. This requires the DNA binding octamer factors Oct-1 and/or Oct-2, as well as an additional B cell-restricted non-DNA binding cofactor. We recently cloned such a coactivator specific for Oct-1 or Oct-2 from human B cells and called it OBF-1. Here we report the isolation and characterization of the murine homologue. Full-length cDNA clones as well as genomic clones were isolated and the gene structure was determined. The deduced protein sequence shows that the mouse protein has an identical length, is likewise proline rich and shows 89% overall identity to the human protein. The OBF-1 gene is expressed in a very highly B cell-specific manner and is transcribed in cells representative of all stages of B cell differentiation, including the earliest ones. We show that OBF-1 interacts in the absence of DNA with the POU domain of Oct-1 or Oct-2 and also with the general transcription factors TBP and TFIIB. Furthermore, we demonstrate that although OBF-1 efficiently activates promoter octamer sites, it does not activate enhancer octamer sites.
A series of 56 azole antifungal agents belonging to chemically diverse families related to bifonazole, one of the antimycotic drugs of clinical use, were investigated using the comparative molecular field analysis (CoMFA) paradigm. The studied compounds, which have been already synthesized and reported to be active in vitro against Candida albicans, were divided into a training set and a test set. The training set consisted of 40 molecules from all the different structural classes. Due to the lack of experimental structural data on these derivatives, molecular mechanics techniques were used to obtain putative active conformations for all the compounds. the correctness of this molecular modeling work was confirmed a posteriori by comparison with structural data of the analog 2w obtained by X-ray crystallographic analysis (Massa, S.; et al. Eur. J. Med. Chem. 1992, 27, 495-502). Two different alignment rules of the training set molecules were used in this study and are based on the assumption that according to published results on azole antifungal agents, all the studied compounds exert their inhibitory activity through the coordination of their azole moiety to the protoporphyrin iron atom of the fungal lanosterol 14alpha-demethylase enzyme. The predictive ability of each resultant CoMFA model was evaluated using a test set consisting of 16 representative compounds that belong to all the different structural classes. The best 3D-quantitative structure-activity relationship model found yields significant cross-validated, conventional, and predictive r2 values equal to 0.57, 0.95, and 0.69, respectively. The average absolute error of predictions of this model is 0.30 log units, and the structural moieties of the studied antifungal agents which are thought to contribute to the biological activity were identified. The predictive capability of this model could be exploited in further synthetic studies on antifungal azoles. Furthermore, the results obtained by using two different alignments of the inhibitors suggest that the binding mode of these molecules involves both a coordination to the iron protoporphyrin atom and an additional, likewise relevant, hydrophobic interaction with the active site.
The synthesis and the evaluation of cytotoxicity and anti-HIV-1 activity of new aryl pyrrolyl (8) and aryl indolyl (9) sulfones are reported. Preparation of above sulfones was achieved by reacting arylsulfonyl chlorides with substituted pyrroles and indoles or by condensing sulfonamides with 2,5-dimethoxytetrahydrofuran in glacial acetic acid according to the Clauson-Kaas method. Chemical requisites relevant to the anti-HIV-1 activity of these compounds are both a 2-sulfonyl-4-chloroanilino moiety and an alkoxycarbonyl group at position 2 of the pyrrole ring. The best activity and selectivity were obtained with ethoxycarbonyl and isopropoxycarbonyl substituents. Substitutions at the amino group of the pharmacophore moiety led to inactive products (alkylation) or weakened (acylation) anti-HIV-1 activity. Among test derivatives, 16 compounds showed EC50 values ranging between 10 and 1 microM, and five (8b',d',f',h'j') showed EC50S in the sub-micromolar range. The compounds were active against HIV-1, both wild type and AZT-resistant strains, but not against HIV-2. Moreover, in enzyme assays they potently inhibited the HIV-1 recombinant reverse transcriptase, were 10 times less active against enzymes from nevirapine- and TIBO-resistant strains, and were totally inactive against the HIV-2 recombinant enzyme. Interestingly, some compounds (8r'-y') were inactive against the recombinant reverse transcriptase while being active in tissue culture.
The amplification of "high" (H) and "low" (L) multispecific antibody responses achieved respectively by H and L lines of selection GP represents a valuable tool in the genetic study of host-infection interactions. These lines were obtained by bidirectional selective breeding for high (HGP) or low (LGP) antibody production to natural complex antigens. HGP and LGP parental lines and reciprocal F1 hybrids, as well as their F2 segregants and backcrosses were submitted to immunization and challenge with rabies virus CVS strain. Acquired resistance was 1000-fold higher in HGP than LGP mice, with a dominance effect to low antibody production observed in F1 hybrids. An association between high antibody response and acquired resistance (P < 0.001) in F2 segregant mice was noticed. The genetic study was performed in these several populations, with a single dose of 104.5-fold LD50 CVS. We could demonstrate 3 independent loci regulating the anti-rabies antibody production, that are distinct, at least in part, from the 10 genes controlling the antigen selection response (sheep erythrocytes) of selection GP.
With the aim of developing novel inhibitors of human immunodeficiency virus, various derivatives (10-17) related to 5H-pyrrolo[1,2-b] [1,2,5]benzothiadiazepine (PBTD) were prepared and tested in vitro. The title tricyclic derivatives were obtained by intramolecular cyclization of the open-chain intermediate arylpyrrylsulfones, followed by N-alkylation at position 10. Among test derivatives some 10-alkyl-5H-pyrrolo[1,2-b] [1,2,5]benzothiadiazepin-11(10H)-one-5,5-dioxides were found to exert potent and specific activity against HIV-1. In particular, 7-chloro derivatives 11i and j showed a potency comparable to that of nevirapine. However, when the chloro atom was shifted to the 8 position, the related products were scarcely active or totally inactive. Replacement of the pyrrole with pyrrolidine led to inactive products and the reduction of SO2 to S strongly diminished the antiviral potency. PBTD derivatives active in cell cultures were also inhibitory to the recombinant HIV-1 RT in enzyme assays, thus allowing the conclusion that PBTDs are a new class of non-nucleoside reverse transcriptase inhibitors (NNRTIs).
A 3-D QSAR study has been carried out on a new class of potent and selective human rhinovirus-14 (HRV-14) inhibitors. In particular, the CoMFA (comparative molecular field analysis) technique has been applied to develop a model able to explain and predict the anti-HRV-14 activity of a class of compounds 4 and potentially helpful to design new and more potent antirhinovirus agents. Docking experiments have also been performed with the aim of elucidating the possible binding mode of these inhibitors. These two approaches are combined to highlight a single, highly favoured mode of interaction of the compounds with the viral capsid proteins.
The synthesis and the in vitro anti-HIV-1 activity of novel pyrrolo annulated benzothiadiazepine acetic acids and some related derivatives are reported. The new compounds share chemical features with pyrrolo[1,2-d][1,4]benzodiazepin-6-one 1 and Ro 5-3335 pyrrylbenzodiazepinone 4, two inhibitors of HIV-replication at the level of reverse transcriptase (RT) and transcriptional transactivation by Tat, respectively. Two derivatives, namely methyl 10,11-dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-acetic-5,5 -dioxide (5a) and 1,12b-dihydro-2H-azeto[2,1-d]pyrrolo[1,2-b][1,2,5]benzoth iadiazepin-2-one 8,8-dioxide (7a), were found to exhibit a significant, although not very potent, activity against human immunodeficiency virus Type 1 (HIV-1).
A new class of potent antifungal agents, namely, 3-aryl-4-[alpha-(1H-imidazol-1-yl)arylmethyl]-pyrroles, is described. These compounds are related to bifonazole and pyrrolnitrin, two compounds belonging to the class of antimycotic drugs. The synthesis of the title pyrroles has been performed starting from 1,3-diaryl-2-propen-1-ones, which were reacted with tosylmethyl isocyanide to give 3-aroyl-4-arylpyrroles. Reduction of the resulting compounds by lithium aluminum hydride furnished the related alcohols, which were treated with 1,1'-carbonyldimidazole to afford the required imidazole derivatives. Forty-four new pyrroles which incorporate an (arylmethyl)imidazole moiety in the 3-arylpyrrole structure were prepared by the above procedure and tested in vitro against Candida albicans and Candida spp. Among test compounds, 10 were found to be highly active against C. albicans. The most active derivative (27) was twice as potent (MIC90) as bifonazole, and its activity was 4 times greater than those of miconazole and ketoconazole. The other nine compounds showed antifungal activity of the same order of that of bifonazole and were ca. 2 times as active as miconazole and ketoconazole. Derivatives 21 and 27 tested in vivo against C. albicans A170 were shown to be highly effective in rabbit skin candidosis. Pharmacological studies on compounds 27 and other related pyrroles (19, 35, 36, 38, 39, and 49) are in progress to select one of them as a potential candidate for clinical experiments.