Search

6-Oxo and 6-thio analogs of purine were prepared based on the initial activity screening of a small, diverse purine library against Mycobacterium tuberculosis (Mtb). Certain 6-oxo and 6-thio-substituted purine analogs described herein showed moderate to good inhibitory activity. N(9)-substitution apparently enhances the anti-mycobacterial activity in the purine series described herein. Several 2-amino and 2-chloro purine analogs were also synthesized that showed moderate inhibitory activity against Mtb.

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf alpha(1-->5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The alpha(1-->5)-linked disaccharides were produced by coupling n-octyl alpha-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H(37)Ra and M. avium complex strains.

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.

The present study extends our previous work regarding new antifolates for Mycobacterium avium (MAC) dihydrofolate reductase (DHFR). The objectives of this study were to synthesize and test new derivatives in the general class of 2,4-diamino-5-methyl-5-deazapteridines in an effort to improve solubility and selectivity for the MAC DHFR, while maintaining lack of selectivity for the human DHFR. New 6-[2', 5'-dialkoxyphenyl) methyl]-substituted DMDP analogs were synthesized as previously described. Three clinical isolates of MAC (NJ211, NJ3404, and NJ168) and M. tuberculosis H37Ra (MTB) were used to evaluate the new derivatives. A previously described colorimetric (alamarBlue(R)) microdilution broth assay was used to determine minimal inhibitory concentrations (MIC). Purified recombinant human (rDHFR), MAC rDHFR, and MTB rDHFR were used in a validated enzyme assay to obtain IC(50) values and to determine selectivity ratios (SR) for the derivatives. For the MAC strains, the MICs ranged from0.25 to16 microg/mL. The most active derivative against MAC was SRI-20920 which had MICs of 0.25, 0.25, and 8 microg/mL for the three strains, respectively. The most selective derivative was SRI-20730 with IC(50s) of 29 and 67,781 nM for MAC rDHFR and hDHFR, respectively, and a SR of 2,337. MICs for MTB ranged from 4 to64 microg/mL and the SR, in general, ranged from 0.32 to 2.5. These results further substantiate the utility of this group of DMDP derivatives for selective activity against MAC.

The preparation of a new 2-carbamoyl pteridine, its activity data against FtsZ from M. tuberculosis (Mtb), and in vitro antibacterial data against Mtb strain H37Ra are presented.

Several octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis ( M.tb. ), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.

Thio analogues of purine, pyridine, and pyrimidine were prepared based on the initial activity screening of several analogues of these heterocycles against Mycobacterium tuberculosis (Mtb). Certain 6-thio-substituted purine analogues described herein showed moderate to good inhibitory activity. In particular, two purine analogues 9-(ethylcarboxymethyl)-6-(decylthio)-9H-purine (20) and 9-(ethylcarboxymethyl)-6-(dodecylthio)-9H-purine (21) exhibited MIC values of 1.56 and 0.78 microg/mL respectively against the Mtb H(37)Rv strain. N(9)-Substitution apparently enhances the antimycobacterial activity in the purine series described herein.

Compounds originally designed as putative tubulin inhibitors were tested as antitubercular agents for inhibition of the Mycobacterium tuberculosis analogue of tubulin, FtsZ. Initial screening of 200 2-alkoxycarbonylpyridines found several that inhibited M. tuberculosis growth. Two compounds, SRI-3072 and SRI-7614, inhibited FtsZ polymerization and were equipotent against susceptible and single-drug-resistant strains of M. tuberculosis. In addition, SRI-3072 reduced the growth of M. tuberculosis in mouse bone marrow macrophages. Our results suggest that these types of compound might be developed into antitubercular drugs effective against the current multidrug-resistant strains of M. tuberculosis.

Analogs of trehalose are reported that were designed to interfere with mycolylation pathways in the mycobacterial cell wall. Several derivatives of 6,6′-dideoxytrehalose, including N , N ′-dialkylamino and 6,6′-bis(sulfonamido) analogs, were prepared and evaluated for antimycobacterial activity against Mycobacterium tuberculosis H 37 Ra and a panel of clinical isolates of Mycobacterium avium . 6,6′-Diaminotrehalose and its diazido precursor were both inactive, but significant activity apparently related to aliphatic chain length was found among the sulfonamides, N -alkylamines, and one of the amidines.

2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium . A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.

Twenty-five 1-deaza-7,8-dihydropteridine derivatives were screened for antimycobacterial activity against Mycobacterium tuberculosis strain H37Ra and three Mycobacterium avium clinical isolates (serovar 1, 4 or 6). Antibacterial activity was determined with a colorimetric microdilution broth assay. Seventeen of the compounds inhibited growth in the range >1.28 to

The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[14C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three β- d -galf(1→4)-α- l -rhamp octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 Download : Download high-res image (126KB) Download : Download full-size image Scheme 1 . Synthesis of disaccharides. Reagents and conditions: (a) Ac2O, Pyridine, rt, 4 h, 98%; (b) SnCl4, CH3(CH2)7OH, CH3CN, 0°C–rt, 83%; (c) NaOMe, MeOH, rt, 2 h, 99%; (d) 2,2′-dimethoxypropane, (1S)-(+)-10-camphorsulphonic acid, acetone, rt, 2 h, 96%; (e) Hg(CN)2, HgBr2, CH3CN, 2 h, 79%; (f) NaOMe, MeOH, rt, 1 h, 90%; (g) TFA containing 1% H2O, CHCl3, rt, 3 h, 91%; (h) TFA containg 1% H2O, CHCl3 rt, 3 h, 85%; (i) NaOMe, MeOH, rt, 1 h, 99%. and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their Km and Vmax values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).

A series of hydrolytically-stable aza analogs of arabinofuranose was prepared and evaluated against Mycobacterium tuberculosis and M. avium. The compounds were designed to mimic the putative arabinose donor involved in biogenesis of the essential cell wall polysaccharide, arabinogalactan. Though most compounds displayed little activity in cell culture, one compound showed significant activity in infected macrophage models.

A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb H(37)Ra and Mtb H(37)Rv are reported. Certain analogs described herein showed moderate to good inhibitory activity.

Herein we report the synthesis of symmetrical C-linked and pseudo-symmetrical O-linked disaccharides structurally related to Araf motifs present in the cell wall of MTB. Their activity in a competition-based arabinosyltransferase assay using [14C]-DPA as the glycosyl donor is also presented. In addition, in vitro inhibitory activity for the disaccharides was determined in a colorimetric broth microdilution assay system against MTB H37Ra and Mycobacterium avium.

Pyranocoumarin compounds were identified to embody a novel and unique pharmacophore for anti-TB activity. A systematic approach was taken to investigate the structural characteristics. Focused libraries of compounds were synthesized and evaluated for their anti-TB activity in primary screening assays. Compounds shown to be active were further determined for MIC and MBC values. Three of the four bactericidal compounds (16, 17c, and 18f) were amino derivatives, with MIC values of 16 microg/mL and respective MBC values of 32, 32, and 64 microg/mL.

Mycobacterium avium complex (MAC) is resistant to trimethoprim, an inhibitor of bacterial dihydrofolate reductase (DHFR). A previously identified selective inhibitor of MAC DHFR, SRI-8858, was shown to have synergistic activity in combination with dapsone and sulfamethoxazole, two drugs that inhibit bacterial dihydropteroate synthase.

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.

The aims of this study were to assess the in vitro activity of 2-methyl-adenosine against Mycobacterium tuberculosis and evaluate, and to intracellular efficacy, and to evaluate its effectiveness against M. tuberculosis in a persistent state model and examine its potential mechanism of action.In vitro activity was determined by means of a colorimetric microdilution broth assay. Intracellular activity was assessed with a Mono Mac 6 human monocytic cell line. A hypoxic shift-down model was used to evaluate the effect of 2-methyl-adenosine on M. tuberculosis in a persistent state. Mechanism-of-action studies were conducted by examining the effect of 2-methyl-adenosine on the uptake of appropriate radiolabelled precursors into respective mycobacterial macromolecular components.Studies confirmed the in vitro activity of 2-methyl-adenosine against M. tuberculosis and demonstrated intracellular efficacy against M. tuberculosis within macrophages. 2-Methyl-adenosine was able to significantly affect the viability of M. tuberculosis in a hypoxic shift-down model previously described to simulate the persistent state that results during tuberculosis. Mechanism-of-action studies revealed that the immediate inhibitory effects of 2-methyl-adenosine were associated with protein and DNA synthesis and not RNA synthesis.Results indicate that 2-methyl-adenosine, or similar derivatives, might be effective against M. tuberculosis infections during latency. This information should be helpful in understanding purine metabolism of M. tuberculosis and also the metabolic activity of this important human pathogen in the persistent state.

A series of pyrazine and quinoxaline derivatives have been synthesized, and their activity against M. tuberculosis (Mtb) and Mycobacterium avium (MAC) are reported. The 4-acetoxybenzyl ester of pyrazinoic acid and 4'-acetoxybenzyl 2-quinoxalinecarboxylate showed excellent activity against Mtb (MIC ranges of less than 1-6.25 microg/mL) but only modest activity against MAC (MICs of 4-32 microg/mL).

The mycobacterial cell wall is a potential target for new drug development. Herein we report the preparation and activity of several n -octyl-5-(α- d -arabinofuranosyl)-β- d -galactofuranoside derivatives. A cell-free assay system has been utilized for determination of the ability of disaccharide analogues to act as arabinosyltransferase acceptors using [ 14 C]-DPA as the glycosyl donor. In addition, in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). One of these disaccharides showed moderate activity against MTB. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to a totally deprotected disaccharide that more closely resembles the natural substrates in cell wall biosynthesis.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[14C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.

Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2,4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168, NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue) microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were also determined. Substitutions at position 5 of the pteridine moiety included -CH 3 , -CH 2 CH 3 , and -CH 2 OCH 3 groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a two-atom bridge of either -CH 2 NH, -CH 2 N(CH 3 ), -CH 2 CH 2 , or -CH 2 S. All but 4 of the 77 derivatives were active against MAC NJ168 at concentrations of ≤13 μg/ml. Depending on the MAC strain used, 81 to 87% had MICs of ≤1.3 μg/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6 and the attached aryl group with substitutions at the 2′ and 5′ positions on the phenyl ring. Using this assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.

Ethambutol is an established front-line agent for the treatment of tuberculosis, and is also active against Mycobacterium avium infection. However, this agent exhibits toxicity, and is considered to have low potency. The action of ethambutol on the mycobacterial cell wall, particularly the arabinan, and comparison of the structure of ethambutol with several of the cell-wall saccharides, suggested that ethambutol–saccharide hybrids might lead to agents with a more selective mechanism of action. To this end, eight ethambutol–saccharide hybrids were synthesized and screened against M . tuberculosis and several clinical isolates of M . avium .

Twelve lipophilic 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim were evaluated for activity against Mycobacterium tuberculosis and Mycobacterium avium in vitro. Six of the compounds had MICs ofor =12.8 mg/L andor =1.28 mg/L against M. tuberculosis and M. avium, respectively; trimethoprim MICs were128 mg/L and12.8 butor =128 mg/L, respectively. Two compounds, with either a 2-methyl-5-methoxy phenyl or 2-methoxy-5-trifluoromethyl phenyl linked at the 6-position of the deazapteridine moiety by a CH2NH bridge, had MICs ofor =0.13 mg/L against M. avium; the two compounds also had apparent I50 values for dihydrofolate reductase of 2 and 8 nM, respectively, compared with an I50 of 400 nM with trimethoprim. Four of the compounds were selectively toxic to mycobacteria as compared with Vero cells. These results demonstrated that lipophilic antifolates can be synthesized which are more active against mycobacteria than trimethoprim and which possess selective toxicity.

Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G + C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence

Lipids of antibiotic-resistant and related -susceptible strains of the Enterobacteriaceae were extracted with chloroform–methanol and characterized by thin-layer chromatography, densitometry, and fatty acid analysis using gas chromatography. Quantitative differences which correlated with antibiotic resistance existed among the phospholipids and fatty acids. A relatively higher concentration of a ninhydrin-positive phospholipid concomitant with a lower amount of phosphatidylethanolamine was observed in antibiotic-resistant strains of Serratia marcescens. Bacterial strains which harbored R-factor 222 had a higher ratio of phosphatidylglycerol to diphosphatidylglycerol than their respective parent strains while those strains which were resistant to the polymyxins had a lower ratio of these phospholipids. Differences in the relative amounts of certain unsaturated and cyclopropane fatty acids were observed between susceptible and resistant strains. Such differences, however, were dependent upon a particular genus and species.

Spirohydantoin mustard (SHM), a central nervous system directed nitrogen mustard with anticancer activity, was metabolized in the presence of mouse liver postmitochondrial supernatant (9000g fraction) to a nonpolar alkylating metabolite. The metabolite was isolated by thin-layer chromatography of chloroform or ethyl acetate extracts of incubation mixtures, and its structure was established by mass spectral analysis, synthesis, and cochromatography. The metabolite, spirohydantoin aziridine, was mutagenic for Salmonella typhimurium TA1535 in the Ames assay but inactive as an antitumor agent against P388 leukemia in vivo.

A microbiologic assay of the agar diffusion type, employing a strain of yeast tentatively identified as Candida macedoniensis, was developed to study the disposition, excretion, and metabolism of the antitumor agent bruceantin (BN; NSC 165563) in the mouse. Bioautographic studies showed the assay to be specific for BN. Normal and tumor-bearing male BDFl mice were studied. Tumor-bearing mice were implanted sc with 10 6 L1210 ascites cells and held for 7 days prior to dosing. Average tumor weight was 307 mg and advanced generalized disease was evident. Three groups of 5 or 10 mice were injected iv with BN (1.5 mg/kg; approx. LD10). At various times after injection, blood, tissues, urine, and feces were obtained and extracted with chloroform to recover BN. Recovery of BN was in the range of 91 to 121%. Disposition and excretion of BN were similar for normal and tumor-bearing mice. Decay of BN in blood was biphasic with α-phase half-lives of 5 to 6 min. Estimated half-lives for the β-phase were possibly >0.5 day. Average zero time intercepts for α-and β-phases were 550 and 51 ng/ml respectively. Higher levels of BN were found in lung, pancreas, intestine, and spleen (1–6 μg/g) than in liver, kidney, and tumor (0.3–0.5 μg/g) after 15 min. Concentrations of BN in brain and peritoneal fat were below detectable limits (0.1 μg/g tissue). Urine and fecal excretion of BN accounted for 2% of the dose after 24 h. In vitro metabolism studies using a postmitochondrial microsome fraction of liver, lung, and kidney suggest that bruceantin is inactivated by an NADPH-dependent enzyme present in liver but not in lung or kidney.

The mechanism for an enhanced effect of Aroclor 1254-induced Sprague-Dawley rat liver 9,000 x g supernatant (S9) microsome preparation on the mutagenicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-N-nitrosourea (CCNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose (chlorozotocin) for Salmonella typhimurium strain TA1535 was studied. Although all three compounds were direct-acting mutagens, rat liver S9 increased the mutagenic response to BCNU, CCNU, and chlorozotocin. The enhanced mutagenic effect was independent of NADPH. Heat-denatured S9 enhanced the mutagenicity of BCNU and CCNU, but not that of chlorozotocin. Mutagenic enhancement, however, was less than that observed with untreated S9. The substitution of extractable S9 lipid and bovine serum albumin for S9 in the reaction mixture resulted in an enhanced mutagenicity of CCNU with little or no effect on BCNU or chlorozotocin mutagenicity. These results suggest that the enhanced mutagenicity of CCNU, and possibly that of BCNU, in the presence of S9 was due in part to nonspecific factors that are present in the S9 preparation.

The effectiveness of surfactants as potentiators of antibiotic activity on several resistant strains of bacteria, selected from clinical sources and laboratory collections, was studied using a tube dilution assay. Bacterial strains included members of the Enterobacteriaceae and staphylococci. Cetyltrimethylammonium bromide (CTAB), Tween 80 (Tw80), a mixture of n -alkyldimethyl betaines (L14), and alpha-(2,4,5-trichlorophenoxy) propionic acid (TCP) were tested in combination with pencillin G (PenG), methicillin (Met), streptomycin (Sm), polymyxin B (PmB), and chlortetracycline (CTC). Growth response to the drug combinations was compared with the response to each drug alone. CTAB and L14 but not Tw80 or TCP were found to potentiate the activity of CTC on strains of Escherichia coli, Proteus mirabilis , and Klebsiella pneumoniae . Studies on the inhibition of protein synthesis by CTC in cells of a strain of E. coli suggested that the surfactants increased the uptake of antibiotic into the cells. CTAB and L14 almost completely sensitized strains of P. mirabilis, Serratia marcescens, K. pneumoniae , and E. coli to PmB. With the exception of K. pneumoniae , TCP was also effective in potentiating the activity of PmB on the above strains whereas Tw80 showed potentiation only with a strain of E. coli . CTAB and L14 but not TCP or Tw80 potentiated the activity of PenG but not Met on strains of staphylococci. Studies of penicillinase in the cells suggested that the surfactants inhibited the formation of this enzyme possibly at the level of induction. None of the surfactants were found to potentiate the activity of Sm.

Glycine-resistant variants of Staphylococcus aureus were obtained by successive cultivation of parent strains in increasing concentrations of glycine, and the minimal inhibitory concentrations of glycine of the parents and variants were determined. Although it has been reported that growth in glycine or certain antibiotics causes the accumulation of nucleotides involved in cell wall synthesis, a lack of cross resistance of the variants to some of these antibiotics was observed.

The metabolic regulation of the synthesis of the folate reductase and the specific dihydrofolate reductase of the amethopterin-resistant bacterium Streptococcus faecium var. durans Ak was investigated. Under growth conditions for maximum synthesis, the differential rate increased two fold during exponential growth. Synthesis of both reductases was coordinate and air-sensitive. The interrelated growth requirements of the bacterium contribute to the complexity of regulation. Folate acts as a “positive” inducer of both types of reductase. Purines and thymine or their metabolic derivatives are interdependent in exerting an action which may antagonize the folate effect. These regulator agents apparently have little selective effect on any of the electrophoretically distinct forms of dihydrofolate reductase. Proportionate synthesis of the folate reductase (the major form), of the specific dihydrofolate reductase (the minor form) and of a minor component of folate reductase occurred in the presence and absence of the exogenous regulators. Amethopterin appears to act as a protective agent of reductase-active protein synthesized during exponential growth.