Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy.
ABSTRACT: There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
Project description:Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids-a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1)?a concise total synthesis of cystobactamid 507, 2)?the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3)?the first structure-activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug-target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.
Project description:QPX7728 is a recently discovered ultra-broad-spectrum beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in beta-lactamase-producing Gram-negative bacteria, including <i>Acinetobacter</i> spp. The potency of meropenem alone and in combination with QPX7728 (1 to 16 μg/ml) was tested against 275 clinical isolates of <i>Acinetobacter baumannii</i> (carbapenem-resistant <i>A. baumannii</i> [CRAB]) collected worldwide that were highly resistant to carbapenems (MIC<sub>50</sub> and MIC<sub>90</sub> for meropenem, 64 and >64 μg/ml). Addition of QPX7728 resulted in a marked concentration-dependent increase in meropenem potency, with the MIC<sub>90</sub> of meropenem alone decreasing from >64 μg/ml to 8 and 4 μg/ml when tested with fixed concentrations of QPX7728 at 4 and 8 μg/ml, respectively. In order to identify the mechanisms that modulate the meropenem-QPX7728 MIC, the whole-genome sequences were determined for 135 isolates with a wide distribution of meropenem-QPX7728 MICs. This panel of strains included 116 strains producing OXA carbapenemases (71 OXA-23, 16 OXA-72, 16 OXA-24, 9 OXA-58, and 4 OXA-239), 5 strains producing NDM-1, one KPC-producing strain, and 13 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 μg/ml). Our analysis indicated that mutated PBP3 (with mutations localized in the vicinity of the substrate/inhibitor binding site) is the main factor that contributes to the reduction of meropenem-QPX7728 potency. Still, >90% of isolates that carried PBP3 mutations remained susceptible to ≤8 μg/ml of meropenem when tested with a fixed 4 to 8 μg/ml of QPX7728. In the absence of PBP3 mutations, the MICs of meropenem tested in combination with 4 to 8 μg/ml of QPX7728 did not exceed 8 μg/ml. In the presence of both PBP3 and efflux mutations, 84.6% of isolates were susceptible to ≤8 μg/ml of meropenem with 4 or 8 μg/ml of QPX7728. The combination of QPX7728 with meropenem against CRAB isolates with multiple resistance mechanisms has an attractive microbiological profile.
Project description:The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 μg mL<sup>-1</sup> against fluoroquinolone-resistant <i>Staphylococcus aureus</i>. Although <i>in vitro</i> hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.
Project description:8-Acetoxycycloberberine (2) with a unique skeleton was first identified to display a potent activity profile against Gram-positive bacteria, especially methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration (MIC) values of 1-8 μg/mL, suggesting a possible novel mechanism of action against bacteria. Taking 2 as the lead, 23 new 8-substituted cycloberberine (CBBR) derivatives including ether, amine, and amide were synthesized and evaluated for their antibacterial effect. The structure-activity relationship revealed that the introduction of a suitable substituent at the 8-position could greatly enhance the potency against MRSA. Among them, compounds 5d and 9e demonstrated equally effective anti-MRSA potency as lead 2, with an advantage of having a more stable pharmacokinetics feature. A preliminary mechanism study indicated that compound 9e acted upon bacteria partly through catalyzing the cleavage of bacterial DNA. Therefore, we consider that 8-substituted CBBR derivatives constitute a promising class of antibacterial agents in the treatment of MRSA infections.
Project description:Filamenting temperature sensitive protein Z (FtsZ) is an essential bacterial cell division protein and a promising target for the development of new antibacterial therapeutics. As a part of our ongoing SAR studies on 2,5,6-trisubstituted benzimidazoles as antitubercular agents targeting <i>Mtb</i>-FtsZ, a new library of compounds with modifications at the 2 position was designed, synthesized and evaluated for their activity against <i>Mtb</i>-H37Rv. This new library of trisubstituted benzimidazoles exhibited MIC values in the range of 0.004-50 μg mL<sup>-1</sup>. Compounds <b>6b</b>, <b>6c</b>, <b>20f</b> and <b>20g</b> showed excellent growth inhibitory activities ranging from 0.004-0.08 μg mL<sup>-1</sup>. This SAR study has led to the discovery of a remarkably potent compound <b>20g</b> (MIC 0.0039 μg mL<sup>-1</sup>; normalized MIC 0.015 μg mL<sup>-1</sup>). Our 3DQSAR model predicted <b>20g</b> as the most potent compound in the library.
Project description:Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a β-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.
Project description:The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50?years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires medium supplemented with Ca<sup>2+</sup>, and the lipoglycopeptides dalbavancin and oritavancin require Tween 80 to be added to the growth medium to prevent the depletion of free drug via adsorption to the plastic microplate. In this report, we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in medium alone and in medium supplemented with the commonly used additives Tween 80, lysed horse blood, and 50% human serum. We observed very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and found that nonspecific binding plates can replace the need for surfactant additives. Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics.
Project description:Seven new coralmycin derivatives, coralmycins C (<b>1</b>), D (<b>2</b>), E (<b>3</b>), F (<b>4</b>), G (<b>5</b>), H (<b>6</b>), and I (<b>7</b>), along with three known compounds, cystobactamids 891-2 (8), 905-2 (9), and 507 (10), were isolated from a large-scale culture of the myxobacteria <i>Corallococcus coralloides</i> M23. The structures of these compounds, including their relative stereochemistries, were elucidated by interpretation of their spectroscopic and CD data. The structure-activity relationships of their antibacterial and DNA gyrase inhibitory activities indicated that the <i>para</i>-nitrobenzoic acid unit is critical for the inhibition of DNA gyrase and bacterial growth, while the nitro moiety of the <i>para</i>-nitrobenzoic acid unit and the isopropyl chain at C-4 could be important for permeability into certain Gram-negative bacteria, including <i>Pseudomonas aeruginosa</i> and <i>Klebsiella pneumoniae</i>, and the ?-methoxyasparagine moiety could affect cellular uptake into all tested bacteria. These results could facilitate the chemical optimization of coralmycins for the treatment of multidrug-resistant Gram-negative bacteria.
Project description:A chemical investigation of the leaves of <i>Tabernaemontana inconspicua</i> Stapf. led to the isolation of a new phenylpropanol derivative, namely irisdichototin G (<b>1</b>), together with nine known compounds, including one polyol derivative, dambonitol (<b>2</b>); three alkaloids, 10-hydroxycoronaridine (<b>3</b>), voacristine (<b>4</b>) and vobasine (<b>5</b>); two triterpenes lupeol (<b>6</b>), betulinic acid (<b>7</b>) and three sterols, sitosterol (<b>8</b>), sitosterol-3-O-β-D-glucopyranoside (<b>9</b>) and stigmasterol (<b>10</b>). The structure of the new compound, as well as those of the known ones, was established by means of spectroscopic methods: NMR analysis (<sup>1H</sup> and <sup>13</sup>C NMR, 1H-1H-COSY, HSQC, HMBC and NOESY), high-resolution mass spectrometry (HR-ESI-MS) and comparisons with previously reported data. Among the known compounds, compound <b>2</b> was firstly reported from the family Apocynaceae. Compounds <b>1</b>-<b>5</b> were tested for their antimicrobial effects against three Gram-negative organisms associated with human wound and systemic infections, namely <i>Haemophilus influenzae</i> 9435337A, <i>Klebsiella pneumoniae</i> 17102005 and <i>Pseudomonas aeruginosa</i> 2137659B. Compounds <b>1</b>, <b>3</b>, and <b>5</b> showed significant antimicrobial effects with minimum inhibitory concentrations (MIC) of 62.5 μg/mL, 62.5 μg/mL and 7.81 μg/mL, respectively, against <i>Haemophilus influenzae</i>, whereas compounds <b>1</b> and <b>5</b> showed significant antimicrobial effects, with a MIC value of 31.25 μg/mL against <i>Pseudomonas aeruginosa</i>. In addition, compound <b>3</b> showed significant antimicrobial activity, with a MIC value of 31.25 μg/mL against <i>Klebsiella pneumoniae</i>.
Project description:There is an urgent need for new therapies to overcome antimicrobial resistance (AMR) especially against Gram-negative bacilli (GNB). Multicomponent therapy combining antibiotics with enhancer molecules known as adjuvants is an emerging strategy to combat AMR. We have previously reported tobramycin-based adjuvants which are able to potentiate various antibiotics. In order to expand the repertoire of tobramycin hybrid adjuvants, a new hybrid containing niclosamide, an FDA approved anthelmintic which has recently demonstrated a variety of interesting biological effects, was synthesized. It was found that this conjugate can potentiate several antibiotics against multidrug-resistant GNB, including the recently approved siderophore cephalosporin cefiderocol. 8 μg ml<sup>-1</sup> of the niclosamide-tobramycin hybrid in combination therapy against a pandrug-resistant strain of <i>P. aeruginosa</i> was able to lower the cefiderocol MIC 32-fold, from 8 μg ml<sup>-1</sup> to 0.25 μg ml<sup>-1</sup> in iron-rich media where siderophore uptake is reduced. These results indicate that a niclosamide-tobramycin hybrid adjuvant can serve to potentiate a newly approved antibiotic.