Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis.

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3 vs CH3) on the central ring oxygen atoms (R(2) and R(3)) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19 b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.5-2 µg/mL). Compounds 29-34 with larger ester and ether groups containing substituted aromatic rings at R(3) exhibited slightly reduced activity (MICs 2-16 µg/mL). One explanation for this attenuated activity could be encroachment of the extended R(3) into the neighboring NADPH co-factor. These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).

Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex.

The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl)-piperidine]palladium(II), allows the final Heck coupling to be performed at 90 °C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140 °C reaction temperature. An analytical protocol for separating the S and R enantiomers of two of the most active compounds is also disclosed. Finally, the X-ray structure for the most active enantiomer of the lead compound, (S)-RAB1, is given.

The structure and competitive substrate inhibition of dihydrofolate reductase from Enterococcus faecalis reveal restrictions to cofactor docking.

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMP(R) sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase.

BACKGROUND: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. METHODS: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K(i) and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. RESULTS: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. CONCLUSIONS: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.

Identification of novel potential antibiotics against Staphylococcus using structure-based drug screening targeting dihydrofolate reductase.

The emergence of multidrug-resistant Staphylococcus aureus (S. aureus) makes the treatment of infectious diseases in hospitals more difficult and increases the mortality of the patients. In this study, we attempted to identify novel potent antibiotic candidate compounds against S. aureus dihydrofolate reductase (saDHFR). We performed three-step in silico structure-based drug screening (SBDS) based on the crystal structure of saDHFR using a 154,118 chemical compound library. We subsequently evaluated whether candidate chemical compounds exhibited inhibitory effects on the growth of the model bacterium: Staphylococcus epidermidis (S. epidermidis). The compound KB1 showed a strong inhibitory effect on the growth of S. epidermidis. Moreover, we rescreened chemical structures similar to KB1 from a 461,397 chemical compound library. Three of the four KB1 analogs (KBS1, KBS3, and KBS4) showed inhibitory effects on the growth of S. epidermidis and enzyme inhibitory effects on saDHFR. We performed structure-activity relationship (SAR) analysis of active chemical compounds and observed a correlative relationship among the IC50 values, interaction residues, and structure scaffolds. In addition, the active chemical compounds (KB1, KBS3, and KBS4) had no inhibitory effects on the growth of model enterobacteria (E. coli BL21 and JM109 strains) and no toxic effects on cultured mammalian cells (MDCK cells). Results obtained from Protein Ligand Interaction Fingerprint (PLIF) and Ligand Interaction (LI) analyses suggested that all of the active compounds exhibited potential inhibitory effects on mutated saDHFR of the drug-resistant strains. The structural and experimental information concerning these novel chemical compounds will likely contribute to the development of new antibiotics for both wild-type and drug-resistant S. aureus.

Synthesis and biological evaluation of 2,4-diaminopyrimidine-based antifolate drugs against Bacillus anthracis.

Due to the innate ability of bacteria to develop resistance to available antibiotics, there is a critical need to develop new agents to treat more resilient strains. As a continuation of our research in this area, we have synthesized a series of racemic 2,4-diaminopyrimidine-based drug candidates, and evaluated them against Bacillus anthracis. The structures are comprised of a 2,4-diaminopyrimidine ring, a 3,4-dimethoxybenzyl ring, and an N-acryloyl-substituted 1,2-dihydrophthalazine ring. Various changes were made at the C1 stereocenter of the dihydrophthalazine moiety in the structure, and the biological activity was assessed by measurement of the MIC and K(i) values to identify the most potent drug candidate.

Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets.

Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an antifolate. The second and third compounds' MOA were not clearly identified, likely because the unique MOA was not represented within the database. The availability of the database generated in this report for Staphylococcus aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but the distinction of unique or broadly acting MOA at this time is less clear.

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase.

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.

Crystal structure of Bacillus anthracis dihydrofolate reductase with the dihydrophthalazine-based trimethoprim derivative RAB1 provides a structural explanation of potency and selectivity.

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (< or =12.8 microg/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P2(1)2(1)2(1), and X-ray diffraction data were collected to a 2.3-A resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.

Establishment of a method for evaluating intracellular antibiotic efficacy in Brucella abortus-infected Mono Mac 6 monocytes.

BACKGROUND: Brucellae produce chronic and often lifelong infections in natural hosts. The persistent nature of these infections is predominantly due to the capacity of these bacteria to maintain intracellular residence in host macrophages. Successful antimicrobial therapy requires eradication of brucellae from this intracellular niche. It is important to seek new and improved antimicrobials for brucellosis therapy as well as a method to efficiently evaluate their intracellular efficacy. OBJECTIVES: For that reason, we have developed a method to evaluate intracellular drug efficacy for new and improved antimicrobials that show initial in vitro activity against Brucella species during drug screening. METHODS: Mono Mac 6 monocytes (MM6) were used because they are the only human cell line that constitutively expresses the phenotypic and functional characteristics of mature monocytes. This cell line has not previously been used with Brucella, therefore parallel studies were performed with J774 murine macrophages. Both cell lines were infected with Brucella abortus 2308 and antibiotics used clinically for treatment of brucellosis were used to determine intracellular efficacy. RESULTS: Significant differences in bacterial burden were observed at or above the MIC in both cell lines. Drug concentrations that fell below the MIC were found to significantly reduce intracellular brucellae only in MM6. CONCLUSIONS: The MM6 intracellular efficacy model will provide a useful method to examine the effect of novel antimicrobials for the treatment of human brucellosis.

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase.

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.

Genetic basis for sulfonamide resistance in Bacillus anthracis.

Natural resistance of field strains of Bacillus anthracis to drugs from the sulfonamide class of antimicrobials that act by inhibiting dihydropteroate synthase (DHPS) has been reported. Though the structure of B. anthracis DHPS has been determined, its connection to the apparent intrinsic sulfonamide resistance of the bacterium has not been established. The aim of this study was to determine if a connection exists between DHPS and the observed sulfonamide resistance of B. anthracis. Microdilution broth assays verified that B. anthracis Sterne is highly resistant to a variety of sulfonamides with minimum inhibitory concentrations (MICs) exceeding 1250 microg/ml. A putative gene encoding DHPS (folP) was amplified from B. anthracis Sterne chromosomal DNA by polymerase chain reaction (PCR) and cloned. Sequence comparisons showed 100% identity with DHPSs from published genome sequences for various strains of B. anthracis. Additionally, expression of folP in B. anthracis Sterne was confirmed. Functionality of the B. anthracis DHPS was confirmed by complementation of an Escherichia coli folP deletion mutant as well as a standard enzyme assay. Concomitant transfer of high level sulfonamide resistance to this mutant along with increased sulfonamide IC(50)values for purified B. anthracis DHPS links DHPS to sulfonamide resistance in B. anthracis. These findings lay the groundwork that will aid future development of antimicrobics that target DHPS to treat anthrax infections.

Efficacy of rifabutin-loaded microspheres for treatment of Mycobacterium avium-infected macrophages and mice.

Two poly(DL-lactide-co-glycolide) microsphere formulations (A, 10% wt/wt, and B, 23% wt/wt, 1-10 microns) were evaluated for intracellular delivery of rifabutin using the J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. Within 7 days, formulation A released 100% in both cell lines and B released 53 and 67% in the J774 and MM6, respectively. Intracellular release of rifabutin with both formulations caused significant reduction of intracellularly replicating Mycobacterium avium (MAC). In MAC-infected beige mice, formulation B (50 mg, intraperitoneal days 0 and 7) completely eliminated infection by 21 days (p < 0.001), similar to a rifabutin daily oral regimen.

Microsphere technology for chemotherapy of mycobacterial infections.

The purpose of this review article is to examine the various studies that have evaluated microspheres for delivery of antimycobacterial drugs. Some of the studies strictly involve the development and evaluation of microspheres for use in antimycobacterial drug delivery, whereas others actually use drug-loaded microspheres to treat mycobacterial infections in cell lines and small animals. Although there is a potential to use microspheres to treat a variety of mycobacterial infections, it appears that most of the studies so far have focused on the etiological agent of tuberculosis, Mycobacterium tuberculosis. As a result, the infectious studies presented here all entail the treatment of that mycobacterial agent. This review will address the following aspects that are important if microspheres are to be considered an acceptable therapeutic tool: 1) in vitro release characteristics, 2) delivery, release and efficacy in macrophages, 3) effectiveness in infected small animal models, 4) safe and combined use with other antimycobacterial agents, and 5) reduced toxicity. It is hoped that once all of these parameters are evaluated, a conclusion regarding the benefit of microsphere technology in the treatment of mycobacterial diseases can be reached.

Metabolism of 2-methyladenosine in Mycobacterium tuberculosis.

2-Methyladenosine (methyl-Ado) has selective activity against Mycobacterium tuberculosis (M. tuberculosis). In an effort to better understand its mechanism of action, we have characterized its metabolism in M. tuberculosis cells. The primary intracellular metabolite of methyl-Ado was 2-methyl-adenylate (methyl-AMP). Very little of the methyl-AMP was metabolized further. A M. tuberculosis strain that was resistant to methyl-Ado did not express adenosine kinase and did not convert methyl-Ado to methyl-AMP in intact cells. In contrast to these results, the primary intracellular metabolite of adenosine in M. tuberculosis cells was ATP, which was readily incorporated into RNA. The rate of metabolism of methyl-Ado to methyl-AMP was similar to the rate of metabolism of adenosine to ATP. Treatment of M. tuberculosis with methyl-Ado did not affect intracellular ATP levels. Methyl-Ado and Ado were also cleaved to 2-methyladenine and adenine, respectively, which accumulated in the medium outside the cells. These studies suggested that methyl-AMP was the active metabolite responsible for the cytotoxicity of this agent. Furthermore, because methyl-Ado was poorly metabolized in human cells, these studies indicated that the selective activity of methyl-Ado was due to its selective activation by M. tuberculosis. These studies have identified two enzyme reactions (Ado kinase and Ado cleavage) in M. tuberculosis that could be exploited for the rational design of new and selective anti-M. tuberculosis agents.

Sustained release characteristics of rifampin-loaded microsphere formulations in nonhuman primates.

Controlled release rifampin-loaded microspheres were evaluated for the first time in nonhuman primates. Animals received either 2.0 g of a large formulation (10-150 microm, 23 wt% rifampin) injected subcutaneously at Day 0 (118-139 mg rifampin/kg), 4.0 g of a small formulation (1-10 microm, 5.8 wt% rifampin) administered intravenously in 2.0 g doses on Day 0 and 7 (62.7-72.5 mg rifampin/kg), or a combination of small and large microspheres (169-210 mg rifampin/kg). Extended rifampin release was observed up to 48 days. Average rifampin concentrations remaining in the liver, lung, and spleen at 30 days were 14.03, 4.09, and 1.98 microg/g tissue, respectively.

Synthesis and biological activity of substituted 2,4-diaminopyrimidines that inhibit Bacillus anthracis.

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 µg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 µg/mL and 0.375-1.5 µg/mL, respectively. It is likely that the S isomers of 1 will bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-1a. The final step in the convergent synthesis of target systems 1 from (±)-1-(1-substituted-2(1H)-phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13) was accomplished via a novel Heck coupling reaction under sealed-tube conditions.