Antibiotics that operate via multiple mechanisms of action are a promising strategy to combat growing resistance. Previous studies have shown that dual action antifolates formed from a pyrroloquinazolinediamine core can inhibit the growth of bacterial pathogens without developing resistance. In this work, we expand the scope of dual action antifolates by repurposing the 2,4-diamino-1,6-dihydro-1,3,5-triazine (DADHT) cycloguanil scaffold to a variety of derivatives designed to inhibit dihydrofolate reductase (DHFR) and disrupt bacterial membranes. Dual mechanism DADHTs have activity against a variety of target pathogens, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa, among other ESKAPEE organisms. Through X-ray crystallography, we confirmed engagement of the Escherichia coli DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in the occluded conformation. Using in vitro inhibition of purified E. coli and Staphylococcus aureus DHFR and disruption of E. coli membranes, we determined that alkyl substitution of dihydrotriazine at the 6-position best optimizes the DADHT's two mechanisms of action. By employing both mechanisms, the DADHT spectrum of activity was extended beyond the scope of traditional antifolates. We are optimistic that the dual mechanism approach, particularly through the action of antifolates, offers a unique means of combating hard-to-treat bacterial infections.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphorylβ-D-ribose 2′-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB. The loss of human lives to tuberculosis (TB) continues essentially unabated as a result of poverty, synergy with the HIV/AIDS pandemic, and the emergence of multidrugand extensively drug-resistant strains of Mycobacterium tuberculosis (1-3). Despite some recent successes, such as the discovery of the diarylquinoline drug TMC207 (4) and the promise of the bicyclic nitroimidazole compounds (5-8), and because of the high attrition rate in drug development (9), much greater effort is required to find better drugs in order to meet the Europe PMC Funders Group Author Manuscript Science. Author manuscript; available in PMC 2011 July 01. Published in final edited form as: Science. 2009 May 8; 324(5928): 801–804. doi:10.1126/science.1171583. Copyright 2009 by the American Association for the Advancement of Science; all rights reserved. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts desired goals of killing persistent tubercle bacilli and reducing TB treatment duration from 6 to less than 3 months (10, 11). A series of sulfur-containing heterocycles was synthesized and tested for antibacterial and antifungal activity (12, 13). Among their derivatives, compounds belonging to the nitrobenzothiazinone (BTZ) class showed particular promise in terms of their potency and specificity for mycobacteria. One of them, 2-[2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (BTZ038), was selected for further studies. This compound (series number 10526038; C17H16F3N3O5S, with a molecular weight of 431.4; logP =2.84) (Fig. 1A) was synthesized in seven steps with a yield of 36%. Structure activity relationship work showed that the sulfur atom and the nitro group at positions 1 and 8, respectively, were critical for activity. BTZ038 has a single chiral center, and both enantiomers, BTZ043 (S) and BTZ044 (R), were found to be equipotent in vitro. Because early metabolic studies with bacteria or mice indicated that the nitro group could be reduced to an amino group, and because many TB drugs are prodrugs that require activation by M. tuberculosis (14), the S and R enantiomers of the amino derivatives and the likely hydroxylamine intermediate were synthesized and tested for antimycobacterial activity in vitro (table S1). The amino (BTZ045, S and R) and hydroxylamine (BTZ046) derivatives were substantially less active (500to 5000-fold). The minimal inhibitory concentrations (MICs) of a variety of BTZs against different mycobacteria were very low, ranging from ~0.1 to 80 ng/ml for fast growers and from 1 to 30 ng/ml for members of the M. tuberculosis complex (13). The MIC of BTZ043 against M. tuberculosis H37Rv and Mycobacterium smegmatis were 1 ng/ml (2.3 nM) and 4 ng/ml (9.2 nM), respectively (Table 1), which compares favorably with those of the existing TB drugs isoniazid (INH) (0.02 to 0.2 μg/ml) and ethambutol (EMB) (1 to 5 μg/ml) (14). From structure activity relationship studies, >30 different BTZ derivatives showed MICs of <50 ng/ml against tubercle bacilli (examples are shown in table S2). Crucially, BTZ043 displayed similar activity against all clinical isolates of M. tuberculosis that were tested, including multidrug-resistant and extensively drug-resistant strains, indicating that it targets a previously unknown biological function (table S3). BTZ043 is bactericidal, reducing viability in vitro by more than 1000-fold in under 72 hours (Fig. 1B), which is comparable to the killing effect seen with INH. In two different model systems (auxotrophy and starvation) involving metabolically inert M. tuberculosis, BTZ043 was less effective, which implies that it blocks a step in active metabolism, similar to INH (14). Observation with time-lapse fluorescence microscopy of individual M. smegmatis cells [expressing green fluorescent protein (GFP)] growing in a microfluidic device (15) revealed that upon exposure to BTZ043, the growth rate decreased rapidly followed by a swelling of the poles and lysis of the cells after a few hours (movie S1). M. tuberculosis showed similar but delayed behavior (movie S2 and fig. S1). Comparative transcriptome analysis of M. tuberculosis offered no evidence of mutagenic or nitrosative gene expression signatures after treatment with BTZ043, although expression of 60 genes was induced (table S4), and this was corroborated with proteomics. The transcriptional signature most resembled that generated by the cell wall inhibitors INH, isoxyl, and ethionamide, with the greatest overlap seen with the response to EMB treatment (16, 17). This is consistent with cell lysis and indicated that BTZ targets cell wall biogenesis. We then tested the uptake, intracellular killing, and potential cytotoxicity of BTZ compounds in an ex vivo model using a high-content screening approach (18, 19) in order to monitor macrophages infected with M. tuberculosis expressing GFP. Macrophages treated Makarov et al. Page 2 Science. Author manuscript; available in PMC 2011 July 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts with BTZ043 were protected (fig. S2) as compared with those treated with the amino derivative BTZ045 or the negative controls [dimethyl sulfoxide (DMSO)]. The deduced MIC of BTZ043 was <10 ng/ml, indicating that this compound is more potent than INH (100 ng/ml) and rifampin (>1 μg/ml) against intracellular bacteria (Fig. 1C). In contrast, the amino metabolite BTZ045 had an MIC of >1μg/ml, which is consistent with the in vitro findings (table S1). As a direct correlate of the antibacterial effect, there was extensive macrophage survival when all compounds were used at doses well above the MIC. BTZ043 was more cytotoxic than INH (Fig. 1C) at the highest concentration tested (10 μg/ml) but nonetheless has a favorable selectivity index of >100. Additional in vitro toxicology tests revealed no particularly unfavorable effects (table S4). The in vivo efficacy of BTZ043 was assessed 4 weeks after a low-dose aerosol infection of BALB/c mice in the chronic model of TB. Four weeks of treatment with BTZ043 reduced the bacterial burden in the lungs and spleens by 1 and 2 logs, respectively, at the concentrations used (Fig. 1D). Additional results suggest that BTZ efficacy is timerather than dose-dependent. Acute (5 g/kg) and chronic (25 and 250 mg/kg) toxicology studies in uninfected mice showed that, even at the highest dose tested, there were no adverse anatomical, behavioral, or physiological effects after one month (table S5). To find the target for BTZ, we employed two independent genetic approaches. First, we identified cosmids bearing DNA from M. smegmatis that confer increased resistance on M. smegmatis, and we pinpointed the region responsible by subcloning. Second, we isolated and characterized mutants of M. smegmatis, M. bovis Bacille Calmette-Guérin (BCG), and M. tuberculosis displaying high-level BTZ resistance. The first approach revealed that the MSMEG_6382 gene of M. smegmatis or its M. tuberculosis ortholog rv3790 mediated increased resistance (Fig. 2A), whereas the second showed that drug-resistant mutants harbor missense mutations in the same gene (Table 1). Biochemical studies showed that rv3790 and the neighboring gene rv3791 code for proteins that act in concert to catalyze the epimerization of decaprenylphosphoryl ribose (DPR) to decaprenylphosphoryl arabinose (DPA) (20), a precursor for arabinan synthesis without which a complete mycobacterial cell wall cannot be produced. These essential membrane-associated enzymes (20-23) have been suggested to act as decaprenylphosphoryl-β-D-ribose oxidase and decaprenylphosphoryl-D-2keto erythro pentose reductase, respectively, and we propose naming them DprE1 and DprE2. In all of the drug-resistant mutants we examined, the same codon of rv3790 (dprE1) was affected, in which Cys387 was replaced by Ser or Gly codons, respectively (Table 1). Mutants, harboring alleles such as those in MN47 or MN84, were rare, arising at a frequency of <10−8, and were dominant over the wild-type gene upon introduction into a BTZ-susceptible mycobacterium. Comparative genomics revealed that the BTZ resistance– determining region of rv3790 was highly conserved in orthologous genes from various actinobacteria, except that in a few cases Cys387 was replaced by Ser or Ala (Fig. 2B). The corresponding bacteria, M. avium and M. aurum, were found to be naturally resistant to BTZ (table S3), thus supporting the identification of DprE1/Rv3790 as the target. Further corroboration was obtained biochemically (Fig. 3) by using membrane preparations from M. smegmatis to catalyze the epimerization reaction from radiolabeled DPR precursor, which was produced in situ from 5-phosphoribose diphosphate (20), in the presence or absence of BTZ. Addition of BTZ038, or its enantiomers BTZ043 and BTZ044, abolished the production of DPA from DPR. This reaction was scarcely affected by either the S or R forms of BTZ045 (Fig. 3A) or by BTZ046 (fig. S3). Using recombinant proteins, we found that the reaction requires both DprE1 and E2 (Rv3790 and Rv3791) (Fig. 3B), with neither enzyme alone capable of catalyzing DPA formation. Furthermore, when BN2, the highly

New or repurposed antibiotics are desperately needed since bacterial resistance has risen to essentially all of our current antibiotics, and few new antibiotics have been developed over the last several decades. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (i.e., β-lactamases) and even induction of efflux mechanisms. Research efforts are described that are designed to determine if the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron chelating compounds called siderophores. Several natural siderophore-antibiotic conjugates (sideromycins) have been discovered and studied. The natural sideromycins consist of an iron binding siderophore linked to a warhead that exerts antibiotic activity once assimilated by targeted bacteria. Inspired these natural conjugates, a combination of chemical syntheses, microbiological and biochemical studies have been used to generate semi-synthetic and totally synthetic sideromycin analogs. The results demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery ("Trojan Horse" antibiotics or sideromycins) and induction of iron limitation/starvation (development of new agents to block iron assimilation). While several examples illustrate that this approach can generate microbe selective antibiotics that are active in vitro, the scope and limitations of this approach, especially related to development of resistance, siderophore based molecular recognition requirements, appropriate linker and drug choices, will be described.

Development of resistance to antibiotics is a major medical problem. One approach to extending the utility of our limited antibiotic arsenal is to repurpose antibiotics by altering their bacterial selectivity. Many antibiotics that are used to treat infections caused by Gram-positive bacteria might be made effective against Gram-negative bacterial infections, if they could circumvent permeability barriers and antibiotic deactivation processes associated with Gram-negative bacteria. Herein, we report that covalent attachment of the normally Gram-positive-only antibiotic, daptomycin, with iron sequestering siderophore mimetics that are recognized by Gram-negative bacteria, provides conjugates that are active against virulent strains of Acinetobacter baumannii, including carbapenemase and cephalosporinase producers. The result is the generation of a new set of antibiotics designed to target bacterial infections that have been designated as being of dire concern.

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.

In order to address the dire need for new antibiotics to treat specific strains of drug resistant Gram-negative bacterial infections, a mixed ligand analog of the natural Acinetobacter baumannii selective siderophore, fimsbactin, was coupled to daptomycin, a Gram-positive only antibiotic. The resulting conjugate 11 has potent activity against multidrug resistant strains of A. baumannii both in vitro and in vivo. The study also indicates that conjugation of siderophores to "drugs" that are much larger than the siderophore (iron transport agent) itself facilitates active uptake that circumvents the normal permeability problems in Gram-negative bacteria. The results demonstrate the ability to extend activity of a normally Gram-positive only antibiotic to create a potent and targeted Gram-negative antibiotic using a bacterial iron transport based sideromycin Trojan horse strategy.

AbstractNitrobenzothiazinone gehören zu den potentesten Antituberkulose‐Wirkstoffen. Hier beschreiben wir eine In‐vivo‐Reduktion der beiden klinischen Kandidaten BTZ043 und PBTZ169, die zur Bildung von Meisenheimer‐Komplexen führt. Diese Reduktion ist reversibel, tritt in allen untersuchten Säugetierspezies auf und hat einen erheblichen Einfluss auf In‐vivo‐ADME‐Charakteristiken und signifikante Auswirkungen auf das Design und die Durchführung von klinischen Studien. Die Reduktion wurde durch chemische Untersuchungen verifiziert, die eine vollständige Charakterisierung des Meisenheimer‐Komplexes und dessen Folgechemie ermöglichten. Die Kombination von In‐vivo‐Studien mit chemischen Arbeiten, wie der LC‐MS‐basierten Charakterisierung und der Assay‐Entwicklung, bildet die Grundlage für eine rationale Leitstrukturoptimierung dieser vielversprechenden Klasse von Antituberkulose‐Wirkstoffen.

Nitrobenzothiazinones are among the most potent antituberculosis agents. Herein, we disclose an unprecedented in vivo reduction process that affords Meisenheimer complexes of the clinical candidates BTZ043 and PBTZ169. The reduction is reversible, occurs in all mammalian species investigated, has a profound influence on the in vivo ADME characteristics, and has considerable implications for the design and implementation of clinical studies. The reduction was confirmed by chemical studies that enabled the complete characterization of the Meisenheimer complex and its subsequent chemistry. Combination of the in vivo and chemical studies with LC-MS characterization and assay development also provides a basis for rational lead optimization of this very promising class of antituberculosis agents.

Nitrobenzothiazinone sind hoch aktive Antibiotika gegen Mycobacterium tuberculosis. Für das Abtöten der Bakterien braucht es die Reduktion der Nitro- zu einer reaktiven Nitrosogruppe. F. Kloss et al. beschreiben in der Zuschrift auf S. 2220 ff. einen bislang übersehenen Pfad der In-vivo-Bioreduktion, bei dem instabile Hydrid-Meisenheimer-Komplexe als Metaboliten auftreten, die durch Luft leicht reoxidiert werden und als Wirkstoffmetaboliten vollkommen neuartig sind.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), is a global public health concern because of the emergence of various resistant strains. Benzothiazin-4-ones (BTZs), represented by BTZ043, are a promising new class of agents for the treatment of tuberculosis and have been shown to kill Mtb in vitro, ex vivo, and in mouse models of TB. Herein we report the design and syntheses of nitroaromatic sulfonamide, reverse-amide, and ester classes of anti-TB agents using a scaffold simplification strategy based on BTZ043. The presented work explores the effect of functional groups such as sulfonamides, reverse-amides, and esters that are attached to the nitroaromatic rings on their anti-TB activity. The in vitro activity of the compounds evaluated against the H37Rv strain of Mtb show that nitroaromatic sulfonamides and nitrobenzoic acid esters with two nitro substituents were most active and highlights the importance of the electronic character (electron deficient aromatic ring) of the nitroaromatic ring as a central theme in these types of nitroaromatic anti-TB agents.

Siderophores are multidentate iron(III) chelators used by bacteria for iron assimilation. Sideromycins, also called siderophore-antibiotic conjugates, are a unique subset of siderophores that enter bacterial cells via siderophore uptake pathways and deliver the toxic antibiotic in a "Trojan horse" fashion. Sideromycins represent a novel antibiotic delivery technology with untapped potential for developing sophisticated microbe-selective antibacterial agents that limit the emergence of bacterial resistance. The chemical synthesis of a series of mono-, bis-, and trihydroxamate sideromycins are described here along with their biological evaluation in antibacterial susceptibility assays. The linear hydroxamate siderophores used for the sideromycins in this study were derived from the ferrioxamine family and inspired by the naturally occurring salmycin sideromycins. The antibacterial agents used were a β-lactam carbacepholosporin, Lorabid, and a fluoroquinolone, ciprofloxacin, chosen for the different locations of their biological targets, the periplasm (extracellular) and the cytoplasm (intracellular). The linear hydroxamate-based sideromycins were selectively toxic toward Gram-positive bacteria, especially Staphylococcus aureus SG511 (MIC = 1.0 μM for the trihydroxamate-fluoroquinolone sideromycin). Siderophore-sideromycin competition assays demonstrated that only the fluoroquinolone sideromycins required membrane transport to reach their cytoplasmic biological target and that a trihydroxamate siderophore backbone was required for protein-mediated active transport of the sideromycins into S. aureus cells via siderophore uptake pathways. This work represents a comprehensive study of linear hydroxamate sideromycins and teaches how to build effective hydroxamate-based sideromycins as Gram-positive selective antibiotic agents.

Amphiphilic star-shaped poly(epsilon-caprolactone)-block-poly(oligo(ethylene glycol) methacrylate) [PCLa-b-POEGMA(b)](4) block copolymers with four arms and varying degrees of polymerization for the core (PCL) and the shell (POEGMA) were used to investigate the solution behavior in dilute aqueous solution using a variety of techniques, including fluorescence and UV/Vis spectroscopy, dynamic light scattering, analytical ultracentrifugation, and isothermal titration calorimetry. Particular emphasis has been applied to prove that the systems form unimolecular micelles for different hydrophilic/lipophilic balances of the employed materials. In vitro cytotoxicity and hemocompatibility have further been investigated to probe the suitability of these structures for in vivo applications. A novel fungicide was included into the hydrophobic core in aqueous media to test their potential as drug delivery systems. After loading, the materials have been shown to release the drug and to provoke therewith an inhibition of the growth of different fungal strains.

A set of 2-benzylsulfanyl derivatives of benzothiazole was synthesized and evaluated for antimicrobial and cytotoxic activities. The biological screening on antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria, yeasts and fungi identified benzylsulfanyl derivatives of benzothiazole as selective inhibitors of mycobacteria. The lead compounds in the set, dinitro derivatives exhibited significant activity against sensitive and multidrug-resistant strains of M. tuberculosis and low cytotoxicity. The QSAR study indicated that the antituberculotic activity is connected with LUMO and HOMO energies. The lower lipophilicity and the increased size of the molecule contribute to antituberculotic activity. Thus, dinitrobenzylsulfanyl derivatives of benzothiazole represent promising small-molecule synthetic antimycobacterials.

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4-(Substituted benzylsulfanyl)pyridine-2-carboxamides 6 were synthesized by a three-step synthesis starting from 4-chloropyridine-2-carboxylic acid and substituted benzyl thiols, with the exception of nitroderivatives. The compounds were evaluated for their anti-TB activity against M. tuberculosis, non-tuberculous mycobacteria (M. kansasii and M. avium), and MDR strains of M. tuberculosis. The activities expressed as the minimum inhibitory concentration (MIC) fall into the range of 8-250 mu mol/L. The substances exhibited similar activities against both sensitive and resistant strains.

1,3-Benzothiazin-4-ones (BTZs) are a novel class of TB drug candidates with potent activity against M. tuberculosis. An in silico ligand-based model based on structure-activity data from 170 BTZ compounds was used to design a new series. Compounds were tested against a panel of mycobacterial strains and were profiled for cytotoxicity, stability, and antiproliferative effects. Several of the compounds showed improved activity against MDR-TB while retaining low toxicity with higher microsomal, metabolic, and plasma stability.

Several novel oxazolidinone antibiotics with a spiropiperazinyl substituent at the 4′-position of the phenyl ring were synthesized through nitroso Diels–Alder chemistry and the in vitro antibacterial activities were evaluated against various Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and mycobacteria (Mycobacterium vaccae, Mycobacterium tuberculosis). Analogs (8a and 12) were active against selected drug resistant microbes, like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and had no mammalian toxicity in a Hep-2 cellular assay (CC50 >100 μM).