Programs to facilitate tuberculosis drug discovery: the tuberculosis antimicrobial acquisition and coordinating facility.

There is a real need to discover new drugs that are active on drug-resistant tuberculosis (TB), and for drugs that will shorten the time of therapy. Large pharmaceutical companies have traditionally led the quest for discovering and developing new antiinfective agents but this is not the case when it comes to diseases like tuberculosis that primarily occur in resource restricted countries. Throughout the world many research groups are actively engaged in the scientific discovery of new TB drugs. Unfortunately, most research laboratories do not have the necessary safety facilities or resources for all facets of TB drug discovery. The Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) was established in order to make comprehensive testing services available at no cost to research laboratories with an interest in discovering new TB drugs. The TAACF is a consortium of contracts managed and funded by the National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, MD) as a resource to support preclinical drug discovery and development. The core of the TAACF is the Southern Research Institute, Birmingham, AL, which supports compound acquisition, storage, medicinal chemistry, and high throughput assays. Other collaborating groups provide biological data on antimycobacterial activity and cytotoxicity, preliminary in vivo toxicity, oral bioavailability and efficacy in animal models, specialty testing (such as activity against non-replicating persistent bacteria), and assistance in technology transfer for developing comprehensive promotional packages and facilitating partnerships with pharmaceutical companies for drug development. The TAACF program and recent progress that has been publicly disclosed by suppliers is reviewed. There are many aspects promising of the program that will not be discussed due to confidentially.

Synthesis of hydrophobic N'-mono and N',N"-double alkylated eremomycins inhibiting the transglycosylation stage of bacterial cell wall biosynthesis.

A series of hydrophobic N'-mono and N',N"-double alkylated derivatives of the glycopeptide antibiotic eremomycin were synthesized by reductive alkylation after preliminary protection of the N-terminal amino group of the peptide backbone. The investigation of the antibacterial activity in vitro showed that N'-C10H21- and N'-p-(p-chlorophenyl)benzyl derivatives of eremomycin are the most active against vancomycin-resistant enterococci among the compounds obtained though they are less effective than the corresponding lipophilic derivatives of vancomycin. The introduction of two hydrophobic substituents led to a decrease in activity against both susceptible and resistant bacteria. The biochemical evaluation of the mode of action revealed that in addition to binding to D-Ala-D-Ala these compounds also have an alternative mechanism of action that does not require substrate binding.

Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

In situ assay for identifying inhibitors of bacterial transglycosylase.

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.

Differential antibacterial activity of moenomycin analogues on gram-positive bacteria.

The moenomycin trisaccharide degradation product and synthetic disaccharide analogues based on the disaccharide core were bactericidal to gram-positive bacteria, inhibited lipid II polymerization, and inhibited cell wall synthesis in Enterococcus faecalis. Truncating moenomycin to the trisaccharide, and building upon the core disaccharide have both led to molecules possessing properties not shared with their respective parent structures.

Antifungal rapamycin analogues with reduced immunosuppressive activity.

Several 1,2,3,4-tetrahydro- and 7-N-hydroxycarbamate derivatives of the natural product rapamycin were prepared and assayed for their immunosuppressive and antifungal profiles. Substitutions at the 7-position indicate the possibility of a differentiated immunosuppressive to antifungal profile, whereas 40-position variants of the tetrahydro-analogues did not show similar differentiated activity.

Inhibition of transglycosylation involved in bacterial peptidoglycan synthesis.

The continuing spectre of resistance to antimicrobial agents has driven a sustained search for new agents that possess activity on drug resistant bacteria. Although several paths are available to reach this goal, the most generalized would be the discovery and clinical development of an agent that acts on a new target which has not yet experienced selective pressure in the clinical setting. Such a target should be essential to the growth and survival of bacteria, and sufficiently different from, or better still non-existent in, the human host. The transglycosylation reaction that polymerizes biochemical intermediates into peptidoglycan qualifies as such a target. This biochemical system accepts the basic unit N-acetylglucosamine-beta-1, 4-N-acetyl-muramyl-pentapeptide-pyrophosphoryl-undecaprenol (lipid II), and leads to polymerization of the N-acetylglucosamine -beta-1, 4-N-acetyl-muramyl-pentapeptide segment into peptidoglycan. Approaches to targeting this reaction include modification of known glycolipid and glycopeptide natural product antibiotics. The synthesis and antibacterial activity of synthetic analogs of moenomycin having novel antibacterial activities not present in the parent structure will be presented, together with the combinatorial chemistry and assay systems leading to their discovery. Likewise, we will discuss chemical modifications to specific glycopeptide antibiotics that have extended their spectrum to include vancomycin resistant enterococci that substitute D-alanyl-D-lactate for D-alanyl-D-alanine in their peptidoglycan. Two differing theories, one positing the generation of high affinity, specific binding to D-alanyl-D-lactate via glycopeptide dimerization and/or membrane anchoring, and the other supporting direct targeting of the modified glycopeptide to the transglycosylation complex, seek to explain the mechanism of action on vancomycin resistant enterococci. Biochemical evidence in support of these two theories will be discussed.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding.

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

Targeting cell wall synthesis and assembly in microbes: similarities and contrasts between bacteria and fungi.

icrobial cells possess a form of exoskeleton called the cell wall that protects the organism from osmotic pressure and environmental insults. Synthesis of the various building blocks that make up the cell wall occurs in the cytoplasm, and thus microbial cells face specific biochemical and biophysical problems related to the polymerization, transport, and assembly of building blocks into the final wall structure at an extra-cellular site. Cell walls must also be metabolically and structurally pliable in order to allow for processes such as repair, secretion, DNA exchange, and cell division. In some cases, bacteria and fungi use similar mechanisms, to accomplish synthesis and assembly, while in other cases each used divergent strategies to accomplish specific functions. This review will summarize recent advances in our understanding of fungal and bacterial cell wall synthesis and assembly. We will compare specific pathways used by both fungi and bacteria, paying particular attention to identifying those areas where what is known in one system may point to approaches to solving unanswered questions in the other. The structure, chemical properties, and mechanism of action of select natural and synthetic products which inhibit synthesis or assembly of cells walls will be discussed in terms of similarities in the structures, and/or steps in the synthetic process targeted. In addition, new targets in the pathways will be presented along with recent approaches to the discovery and design of novel inhibitors.

Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

Cellular accumulation, localization, and activity of a synthetic cyclopeptamine in fungi.

A novel synthetic cyclopeptamine, A172013, rapidly accumulated by passive diffusion into Candida albicans CCH442. Drug influx could not be totally facilitated by the membrane-bound target, beta-(1,3)-glucan synthase, since accumulation was unsaturable at drug concentrations up to 10 microg/ml (about 1.6 x 10(-7) molecules/cell), or 25x MIC. About 55 and 23% of the cell-incorporated drug was associated with the cell wall and protoplasts, respectively. Isolated microsomes contained 95% of the protoplast-associated drug, which was fully active against glucan synthesis in vitro. Drug (0.1 microg/ml) accumulation was rapid and complete after 5 min in several fungi tested, including a lipopeptide/cyclopeptamine-resistant strain of C. albicans (LP3-1). The compound penetrated to comparable levels in both yeast and hyphal forms of C. albicans, and accumulation in Aspergillus niger was 20% that in C. albicans. These data indicated that drug-cell interactions were driven by the amphiphilic nature of the compound and that the cell wall served as a major drug reservoir.

Identification and kinetic analysis of a functional homolog of elongation factor 3, YEF3 in Saccharomyces cerevisiae.

Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nM). K(m) values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/K(m) values for EF-3A were about two-fold higher; however, the difference in Kcat/K(m) values between the two factors was small for basal ATPase activity.

Molecular basis of clarithromycin activity against Mycobacterium avium and Mycobacterium smegmatis.

Clarithromycin, the 6-O-methyl derivative of erythromycin, is approved for treatment of Mycobacterium avium infections and for prophylaxis in patients at risk. Since clarithromycin is more active against mycobacteria than the parent compound, erythromycin, we evaluated the interaction of erythromycin and clarithromycin with cells and ribosomes isolated from M. avium and Mycobacterium smegmatis. The MIC of clarithromycin was 32 and 64 times lower than that of erythromycin for M. smegmatis and M. avium, respectively. The cellular uptake rate for clarithromycin was two- to five-fold faster than for erythromycin, and cell-associated clarithromycin reached a plateau two-fold higher than that of erythromycin after 3 h. Energy was not required for uptake. Fractionation of cell-associated clarithromycin yielded 12% in the walls, 21% bound to ribosomes, with the remainder being lost during work-up. In addition, three- to six-fold more clarithromycin was associated with the isolated cell integument compared with erythromycin. The Kd for clarithromycin binding to ribosomes was 2.9- and 3.5-fold tighter for M. smegmatis and M. avium, respectively, than for erythromycin, due mainly to a slower off-rate. The log partition coefficients of the non-ionized form (log Pu) for clarithromycin and erythromycin were 3.24 and 2.92, respectively. Thus clarithromycin is more hydrophobic than erythromycin. This would favour more rapid diffusion within and across hydrophobic regions of the cell integument, since once a solute saturates a membrane the net flux across the membrane must equal the net flux within the membrane as dictated by diffusion. We conclude that the lower MIC of clarithromycin for M. avium and M. smegmatis is due to a combination of increased cellular uptake, the major factor, possibly through a peripheral hydrophobic layer, and increased binding affinity to ribosomes.

Novel antifungal agents which inhibit lanosterol 14alpha-demethylase in Candida albicans CCH442.

We have identified four non-azole inhibitors of lanosterol 14a-demethylase in Candida albicans CCH442. The most potent compound, A-39806, had IC50 values for ergosterol inhibition of 0.9 microM (0.3 mg/L) and 1.9 microM (0.6 mg/L) in whole cell and cell-free extract assays, respectively. A-39806 demonstrated broad in-vitro antifungal activity against several Candida species as well as against Cryptococcus albidus and Aspergillus niger. In-vitro antifungal activity was also demonstrated against a fluconazole-resistant clinical isolate of C. albicans.

Characterization of a lipopeptide-resistant strain of Candida albicans.

Lipopeptides are antifungal agents that inhibit cell wall beta-(1,3)-glucan biosynthesis in fungal organisms. A mutant resistant to lipopeptides was generated by UV mutagenesis and characterized. The Candida albicans mutant (LP3-1) was stable and showed resistance specificity to a broad range of lipopeptides and certain glycolipid inhibitors. Other antifungal agents with diverse modes of action had a normal minimum inhibitory concentration profile for LP3-1 compared with the wild-type strain (CCH 442). In the in vitro beta-(1,3)-glucan synthase assay, both the lipopeptides and papulacandin-related agents had considerably higher 50% inhibitory concentration values in the LP3-1 strain than in the wild-type strain. In reconstitution assays, the resistance factor was associated with the integral membrane pellet rather than the peripheral GTP-binding protein. The LP3-1 strain had a membrane lipid profile similar to that of the parent strain and was virulent in a murine model of systemic candidiasis. Taken together, these results indicate that the resistance factor is associated with the integral membrane component of beta-(1,3)-glucan synthase. Lipopeptides are common antifungal agents encountered during screening of natural products. The LP3-1 strain was resistant to natural product extracts known to contain various lipopeptides. Thus, LP3-1 can be used in a dereplication assay.

Inhibition of 2,3-oxidosqualene-lanosterol cyclase in Candida albicans by pyridinium ion-based inhibitors.

The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpyridinium and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpicolinium cations were evaluated for their ability to inhibit 2,3-oxidosqualene-lanosterol cyclase activity in Candida albicans. Both compounds inhibited fungal growth, were fungicidal, and resulted in the accumulation of squalene epoxide concurrent with a decrease in ergosterol, monomethyl sterols, and lanosterol, as was expected for the specific inhibition of 2,3-oxidosqualene-lanosterol cyclase activity. These compounds are electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene, which may explain their selective action.

Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18).

Zymosan, the cell wall from Saccharomyces cerevisiae, was reported to be a macrophage activator through its beta-glucan over 30 yr ago. Nevertheless, the identity of the beta-glucan receptor has been controversial. This study showed that the alpha M beta 2-integrin, CR3 (Mac-1, CD11b/CD18) served as the beta-glucan receptor through one or more lectin sites located outside of the CD11b I-domain that contains the binding sites for iC3b, ICAM-1, and fibrinogen. Sugar specificity, analyzed with FITC-labeled soluble polysaccharides and flow cytometry, showed CR3-specific staining with several pure beta-glucans but not with alpha-mannan. However, a 10-kDa soluble zymosan polysaccharide (SZP) with high affinity (6.7 x 10(-8) M) for CR3 consisted largely of mannose and approximately 5% glucose. Binding of either SZP-FITC or beta-glucan-FITC to CR3 was blocked not only by pure beta-glucans from yeast, mushroom, seaweed, or barley, but also by N-acetyl-D-glucosamine (NADG), alpha- or beta-methylmannoside, and alpha- or beta-methyl-glucoside. SZP-FITC and beta-glucan-FITC stained all leukocyte types similarly to anti-CR3-FITC, and polysaccharide-FITC staining was inhibited > or = 95% by unlabeled anti-CR3. SZP-FITC staining of cells expressing recombinant chimeras between CR3 and CR4 (p150,95, CD11c/CD18) suggested that both the divalent cation-binding region of CD11b and the region C-terminal to it may regulate binding of polysaccharides to CR3. Unlabeled SZP or beta-glucan also blocked CR3 staining by 11 mAb to C-terminal domain epitopes of CD11b but had no effect on staining by mAb directed to the I-domain. In conclusion, CR3 serves as the leukocyte beta-glucan receptor through a cation-independent lectin site located C-terminal to the I-domain of CD11b. Its sugar specificity is broader than originally appreciated, allowing it to react with certain polysaccharides containing mannose or NADG, as well as glucose.

Design, synthesis and in vitro evaluation of pyridinium ion based cyclase inhibitors and antifungal agents.

The design, synthesis and in vitro biological evaluation of pyridinium ion based inhibitors of oxidosqualene cyclase enzymes are reported. N-Alkyl- and N-prenylpyridinium ions have been found to be potent and specific inhibitors of Candida albicans oxidosqualene-lanosterol cyclase and to exhibit antifungal activity. The ability of pyridinium ions to inhibit the C. albicans cyclase increases with increasing structural resemblance to a putative monocyclized species formed during the course of the cyclization process. The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpyridinium cation 1 inhibits the C. albicans enzyme at concentrations more than 100-fold lower than does the directly analogous piperidinium derivative 4.