Skin permeability of various non-steroidal anti-inflammatory drugs in man

The skin permeabilities of a series of eight salicylates and ten non-steroidal anti-inflammatory drugs were investigated in human subjects. The logarithms of % absorption through intact skin and of n-octanol/water partition coefficients (log P) of the test compounds were plotted against one another, and a parabolic relationship was obtained in both compound series.     

New C25 carbamate rifamycin derivatives are resistant to inactivation by ADP-ribosyl transferases

A novel series of 3-morpholino rifamycins in which the C25 acetate group was replaced by a carbamate group were prepared and found to exhibit significantly improved antimicrobial activity than rifampin against Mycobacterium smegmatis. Further characterization of such compounds suggests that relatively large groups attached to the rifamycin core via a C25 carbamate linkage prevent inactivation via ribosylation of the C23 alcohol as catalyzed by the endogenous rifampin ADP-ribosyl transferase of M. smegmatis. SAR studies of the C25 carbamate rifamycin series against M. smegmatis and other bacteria are reported.     

Synthesis and antimicrobial activity of N¹-benzyl or N¹-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamines

The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N(1)-benzyl and N(1)-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献   

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.     

Quantitative analysis of uncoupling activity of substituted phenols with a physicochemical substituent and molecular parameters

The uncoupling potency of a series of substituted phenols with rat-liver mitochondria was analyzed quantitatively with physicochemical substituent and molecular parameters such as log P, P being the partition coefficient in a phosphatidylcholine liposome/water system, log KA, KA being the acid dissociation constant, and the Taft-Kutter-Hansch steric constant, Es, for ortho-substituents. The potency evaluated from the concentration in the medium required for a defined response was analyzed, showing that the incorporation of compounds in terms of log P, a certain balance between neutral and ionized forms expressible by a parabolic function of log KA and the steric shielding effect of the ortho-substituents on the negatively charged center of ionized form are highly significant factors governing the variations in potency. The potency was also quantitatively separated into the intrinsic potency as the protonophore inside the inner mitochondrial membrane and the incorporation factor in terms of log P. Some phenols found as outliers from the correlations and some others distorting the quality of the correlations were shown to have inhibitory effects on the respiratory chain by specific and non-specific modes of action, respectively, besides uncoupling activity.     

Sulphatide metabolism in brain.

Octanol-water partition coefficients (log P) were determined for a series of substituted psychotomimetic phenethylamine derivatives. A relationship was established between log P, steric bulk in the paraposition and the ability to stimulate serotonin (5-HT) receptors in an $\text{in vitro}$ sheep umbilical artery preparation. It appears that Log P values and $\text{in vitro}$ activity in this preparation.may be useful in predicting hallucinogenic potency in man.     

Antimicrobial activity and physico-chemical properties of some alkyldimethylbenzylammonium chlorides

A series of alkyldimethylbenzylammonium chlorides have been synthesized with n-alkyl chain lengths of C1 leads to C18. Octanol/water partition coefficients were determined and the antimicrobial activity assessed as the minimum growth inhibitory concentrations towards twelve strains of micro-organisms, representative of Gram-negative and Gram-positive bacteria, yeasts and fungi. The data were subjected to a numerical analysis. Antimicrobial activity of the compounds was found to be a parabolic function of their lipophilicity and maximized with n-alkyl chain lengths of between C12 and C16. The data fit to quadratic functions estimated for low (C1-C7) and high (C8-C16) alkyl chain length compounds was better than for a single quadratic describing the activity of the complete series (C1-C18). These maximized at log P values corresponding to alkyl-chain lengths of approximately C7 and C14 respectively, and were suggestive of low and high affinity binding sites upon the cell surface. The data analysis allowed the chain lengths of compounds with optimal activity towards the various groups of organisms to be determined. Generally yeasts and fungi were most sensitive towards C12, Gram-positive bacteria towards C14, and the Gram-negative bacteria towards C16. Gram-negative cells were the most resistant towards all the compounds and Gram-positive cells the least.     

Comparative in vitro and in vivo studies on long-wavelength photosensitizers derived from bacteriopurpurinimide and Bacteriochlorin p6: fused imide ring enhances the in vivo PDT efficacy

In situ conversion of bacteriochlorophyll-a, present in Rhodobacter sphaeroides (Rb. sphaeroides) gave bacteriopurpurin-18 in modest yield, which in a sequence of reactions was converted into two series of bacteriochlorins: bacteriopurpurinimide and bacteriopurpurin p6 with and without a fused imide ring system, respectively. To determine the effect of overall lipophilicity in photosensitizing efficacy, these bacteriochlorins were independently reacted with HBr gas and subsequently treated with various alkyl alcohols to afford the corresponding alkyl ether derivatives as diastereomeric mixtures (the R- and S-isomers were obtained in almost equal ratios). Between the two series of bacteriochlorins, the bacteriopurpurinimides containing a fused imide ring system were found to be more effective in vivo (C3H mice bearing RIF tumors). To investigate the effect of the presence of the chiral center at position 3 of the most effective purpurinimide 9 [3(1'-heptyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-N-hexylimide propyl ester], the acetyl group was replaced with a hydroxymethyl substituent and converted into 3(1'-decyloxy)methyl-3-deacetyl-purpurin-18-N-hexylimide methyl ester 26 with a similar lipophilicity. Interestingly, compared to 26, the bacteriopurpurinimide 9 was found to be more effective, suggesting that the chiral center at position 3 certainly plays an important role in photosensitizing activity. Among a series of alkyl ether analogues, between the PDT efficacy and the lipophilicity (log P and log D) calculated by computational methods (PALLAS program), a parabolic relationship was observed to some extent. However, it was limited to a particular series, e.g., compounds with similar log P values between bacteriopurpurinimides and bacteriochlorin e6 did not produce similar in vivo efficacy. As expected, within a series, a linear relationship was observed between the log P values and the HPLC retention times of the photosensitizers. Some of the mitochondrial localized photosensitizers showed a significant peripheral benzodiazepine binding (PBR) affinity. However, limited correlation between PBR binding affinity and in vivo PDT efficacy was observed. Compared to the naturally occurring bacteriochlorophyll-a, the bacteriopurpurinimides with fused imide ring system showed higher in vitro/in vivo stability. In contrast to methyl pyropheophorbide-a, the ester functionalities in bacteriopurpurinimide did not convert into the corresponding carboxylic acid by the enzyme esterases.     

Porins are required for uptake of phosphates by Mycobacterium smegmatis

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.     

Synthesis, biological activity, QSAR and QSPR study of 2-aminobenzimidazole derivatives as potent H3-antagonists

We report the design, synthesis, QSPR and QSAR of a new class of H(3)-antagonists, having a 2-aminobenzimidazole moiety connected to the 4(5) position of an imidazole ring through di- or tri-methylene chains. Eleven substituents, selected by experimental design to obtain broad and non-correlated variation in their lipophilic, electronic and steric properties, were introduced at the 5(6) position of the benzimidazole nucleus. The compounds were tested for their H(3)-receptor affinity, by displacement of [(3)H]-(R)-alpha-methylhistamine ([(3)H]-RAMHA) binding to rat brain membranes (pK(i)), for intrinsic activity, evaluating their effect on [(35)S]GTPgammaS binding to rat brain membranes, and for H(3)-antagonist potency, on electrically stimulated guinea-pig ileum (pK(B)). The pK(i) values of the derivatives with longer chain (5a-k) ranged over 2 orders of magnitude, with the 5(6)-methoxy derivative 5d endowed with sub-nanomolar affinity (pK(i)=9.37). The series having two methylene groups in the chain spacer (4a-k), showing a small variation in affinity, revealed to be somewhat insensitive to ring substitution. Lipophilicity (log P) and basicity (pK(a)) of the newly synthesized compounds were measured and related to receptor affinity in a QSAR study. Multiple regression analysis (MRA) showed an approximate parabolic dependence of pK(i) on log P, while an additional electronic effect of the substituents on benzimidazole tautomerism is suspected.     

The measurement of adenylate energy charge (AEC) in mycobacteria, including Mycobacterium leprae

Abstract The adenylate energy charge (AEC) of Mycobacterium leprae, Mycobacterium lepraemurium and the cultivable Mycobacterium smegmatis were determined following incubation in a variety of culture conditions. The AEC values for M. smegmatis were similar to those reported for other cultivable bacteria. The AEC values for M. leprae and M. lepraemurium purified from host tissue were lower than those of in vitro-grown organisms. The possible use of the AEC in in vitro studies with M. leprae is discussed.     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Synthesis and antiamoebic activity of new oxime ether derivatives containing 2-acetylpyridine/2-acetylfuran

Various oxime ether derivatives of 2-acetylpyridine and 2-acetylfuran series have been synthesised. O-Alkylation of the oximes by various alkylaminoethyl halides gave the corresponding oxime ether derivatives. The structures of these compounds were elucidated by UV, IR, 1H NMR, 13C NMR spectroscopic methods and elemental analyses. All the compounds were screened in vitro against the HM1:IMSS strain of Entamoeba histolytica. Based on the 50% inhibitory concentration (IC50) data of the 12 compounds evaluated, two of the 2-acetylpyridine series and two in the 2-acetylfuran series showed better IC50 values in vitro when compared with the standard amoebicidal drug, metronidazole. Moreover, one compound showed the most promising antiamoebic activity (IC50=0.5 microM vs IC50=1.9 microM of metronidazole).     

An assessment of two Carpobrotus species extracts as potential antimicrobial agents.

For centuries, indigenous people in South Africa have used a variety of medicinal herbs to treat chronic infections. This investigation focused on two Carpobrotus species belonging to the family, Aizoaceae, in an attempt to assess their antimicrobial potential. Extracts of varying polarities of the plants were prepared and tested against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Mycobacterium smegmatis. For the disc diffusion method, Ciprofloxacin (40 microg/disc) served as positive control for S. aureus, P. aeruginosa and M. smegmatis, whereas amphotericin B (25 microg/disc) was the control for C. albicans. A sample concentration of 10 mg/ml was used. Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution. Phytochemical analysis was completed to test for the presence of flavonoids, hydrolysable tannins, phytosterols and aromatic acids. The ethyl acetate extracts (21 microl of 95 mg/ml) were used for bio-autography, together with TLC analyses. Carpobrotus muirii and Carpobrotus quadrifidus showed antimicrobial activity against S. aureus and M. smegmatis in the disc diffusion method and inhibition against S. aureus and M. smegmatis was observed by clear zones on the TLC plate. This investigation confirms that extracts of these Carpobrotus species that are used as indigenous medicines, exhibit anti-bacterial activity. This scientific information can serve as an important platform for the development of inexpensive, safe and effective natural anti-infective medicines.     

Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides

Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells . The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides.     

Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis

Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.     

Isothiocyanates. A new class of uncouplers.

This paper describes the uncoupling effect of three isothiocyanates: p-bromophenylisothiocyanate, 4,4'-diisothiocyanatebiphenyl and beta-naphtylemthylisothiocyanate on the respiration of Ehrlich-Lettré cells and isolated mitochondria. The isothiocyanates are similar to other uncouplers (such as 2,4-dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone) in that they: 1. stimulate respiration of state 4 mitochondria; 2. stimulate mitochondrial ATPase activity; 3. release the inhibition of mitochondrial respiration by oligomycin and 4. inhibit both mitochondrial respiration and mitochondrial ATPase activity at higher molar concentrations. The incoupling activity of these isothiocyanates correlates well with their biological activity. Maximal activation of a latent mitochondrial ATPase activity of rat liver mitochondria in the presence of p-bromophenylisothiocyanate was found at a concentration of 15 muM. The investigated isothiocyanates differ significantly in their solubility in organic solvents and their chemical reactivity. We assume that the greater the partition coefficient in a series of isothiocyanates grouped according to the increasing value of log P (partition coefficient for the system octanol/water, 25 degrees C), the greater will be their uncoupling activity, but only up to a certain degree. Any further increase of log P will be marked by a decrease of this activity.