An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it.

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion.

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

A method for tritiation of iboxamycin permits measurement of its ribosomal binding.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).

Proposed Resolution of a Mechanistic Puzzle of Long Duration: Self-Condensation of Cyclopentanone to Form an 11-Carbon Dienoic Acid.

The mechanism proposed for the transformation of cyclopentanone to the dienoic acid 1, as published in this journal, is revealed to be in error. We show that carbon 11 derives not from dimethyl sulfoxide as proposed but from the dichloromethane present in the "quenching" solution. The intermediacy of an α-chloromethyl ketone and its subsequent fragmentation in the presence of a hydroxide ion is supported by experiments described herein and by extensive literature precedent.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

[3+2] Dipolar Cycloaddition of a Stabilized Azomethine Ylide and an Electron-Deficient Dipolarophile: Revision of Regioselectivity.

The regioselectivity of a [3+2] dipolar cycloaddition reaction of a stabilized azomethine ylide with an electron-deficient dipolarophile was found to be counter to a report published in this journal.

Synthetic oxepanoprolinamide iboxamycin is active against Listeria monocytogenes despite the intrinsic resistance mediated by VgaL/Lmo0919 ABCF ATPase.

Background: Listeriosis is a food-borne disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes. Clinical L. monocytogenes isolates are often resistant to clinically used lincosamide clindamycin, thus excluding clindamycin as a viable treatment option. Objectives: We have established newly developed lincosamide iboxamycin as a potential novel antilisterial agent. Methods: We determined MICs of the lincosamides lincomycin, clindamycin and iboxamycin for L. monocytogenes, Enterococcus faecalis and Bacillus subtilis strains expressing synergetic antibiotic resistance determinants: ABCF ATPases that directly displace antibiotics from the ribosome and Cfr, a 23S rRNA methyltransferase that compromises antibiotic binding. For L. monocytogenes strains, either expressing VgaL/Lmo0919 or lacking the resistance factor, we performed time-kill kinetics and post-antibiotic effect assays. Results: We show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919 ABCF ATPase. While iboxamycin is not bactericidal against L. monocytogenes, it displays a pronounced post-antibiotic effect, which is a valuable pharmacokinetic feature. We demonstrate that VmlR ABCF of B. subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of E. faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr, resulting in up to a 512-fold increase in MIC. Conclusions: While iboxamycin is a promising new antilisterial agent, our findings suggest that emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored.

Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

A synthetic antibiotic class overcoming bacterial multidrug resistance.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.

Practical Gram-Scale Synthesis of Iboxamycin, a Potent Antibiotic Candidate.

A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is presented. A pivotal transformation in the route involves an intramolecular hydrosilylation-oxidation sequence to set the ring-fusion stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective alkylation of a pseudoephenamine amide, a convergent sp3-sp2 Negishi coupling, and a one-pot transacetalization-reduction reaction to form the target compound's oxepane ring. Implementation of this synthetic strategy has provided ample quantities of iboxamycin to allow for its in vivo profiling in murine models of infection.

A Practical, Component-Based Synthetic Route to Methylthiolincosamine Permitting Facile Northern-Half Diversification of Lincosamide Antibiotics.

The development of a flexible, component-based synthetic route to the amino sugar fragment of the lincosamide antibiotics is described. This route hinges on the application and extension of nitroaldol chemistry to forge strategic bonds within complex amino sugar targets and employs a glycal epoxide as a versatile glycosyl donor for the installation of anomeric groups. Through building-block exchange and late-stage functionalization, this route affords access to a host of rationally designed lincosamides otherwise inaccessible by semisynthesis and underpins a platform for the discovery of new lincosamide antibiotics.

Discovery of Macrolide Antibiotics Effective against Multi-Drug Resistant Gram-Negative Pathogens.

Macrolides are among the most widely prescribed antibiotics, particularly for bacterial lung infections, due to their favorable safety, oral bioavailability, and spectrum of activity against Gram-positive pathogens such as Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Their utility against Gram-negative bacteria is extremely limited and does not include the Enterobacteriaceae or other ESKAPE pathogens. With the increasing development of resistance to current therapies and the lack of safe, oral options to treat Gram-negative infections, extended-spectrum macrolides have the potential to provide valuable treatment options. While the bacterial ribosome, the target of macrolides, is highly conserved across Gram-positive and Gram-negative bacteria, traditional macrolides do not possess the proper physicochemical properties to cross the polar Gram-negative outer membrane and are highly susceptible to efflux. As with most natural product-derived compounds, macrolides are generally prepared through semisynthesis, which is limited in scope and lacks the ability to make the drastic physicochemical property changes necessary to overcome these hurdles.By using a fully synthetic platform technology to greatly expand structural diversity, novel macrolides were prepared with a focus on lowering the MW and increasing the polarity to achieve a physicochemical property profile more similar to that of traditional Gram-negative drug classes. In addition to the removal of lipophilic groups, a critical structural feature for obtaining Gram-negative activity in the macrolide class proved to be the introduction of small secondary or tertiary amines to yield polycationic species potentially capable of self-promoted uptake. Within the azithromycin-like 15-membered azalides, potent activity was seen when small alkyl amines were introduced at the 6'-position of desosamine. The biggest gains, however, were made by replacing the entire C10-C13 fragment of the macrolactone ring with commercially available or readily synthesized 1,2-aminoalcohols, leading to 13-membered azalides. The introduction of a tethered basic amine at the C10-position and systematic optimization of substitution and tether length and flexibility ultimately provided new macrolides that for the first time exhibit clinically relevant antibacterial activity against multi-drug resistant Gram-negative bacteria. A retrospective computational analysis of >1800 fully synthetic macrolides prepared during this effort identified key drivers and optimum ranges for improving permeability and avoiding efflux. In contrast to standard Gram-negative drugs which generally have MWs below 600 and clogD7.4 values below 0, we found that the ideal ranges for Gram-negative macrolides were MW between 600 and 720 and cLogD7.4 between -1 and 3. A total charge of between 2.5 and 3 was also required to provide optimal permeability and efflux avoidance. Thus, Gram-negative macrolides occupy a unique physicochemical property space that lies between traditional Gram-negative drug classes and Gram-positive macrolides.

Tetracyclines promote survival and fitness in mitochondrial disease models.

Mitochondrial diseases (MDs) are a heterogeneous group of disorders resulting from mutations in nuclear or mitochondrial DNA genes encoding mitochondrial proteins1,2. MDs cause pathologies with severe tissue damage and ultimately death3,4. There are no cures for MDs and current treatments are only palliative5-7. Here we show that tetracyclines improve fitness of cultured MD cells and ameliorate disease in a mouse model of Leigh syndrome. To identify small molecules that prevent cellular damage and death under nutrient stress conditions, we conduct a chemical high-throughput screen with cells carrying human MD mutations and discover a series of antibiotics that maintain survival of various MD cells. We subsequently show that a sub-library of tetracycline analogues, including doxycycline, rescues cell death and inflammatory signatures in mutant cells through partial and selective inhibition of mitochondrial translation, resulting in an ATF4-independent mitohormetic response. Doxycycline treatment strongly promotes fitness and survival of Ndufs4-/- mice, a preclinical Leigh syndrome mouse model8. A proteomic analysis of brain tissue reveals that doxycycline treatment largely prevents neuronal death and the accumulation of neuroimmune and inflammatory proteins in Ndufs4-/- mice, indicating a potential causal role for these proteins in the brain pathology. Our findings suggest that tetracyclines deserve further evaluation as potential drugs for the treatment of MDs.

Tetracyclines Modify Translation by Targeting Key Human rRNA Substructures.

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

Large-scale preparation of key building blocks for the manufacture of fully synthetic macrolide antibiotics.

Key building blocks for the production of fully synthetic macrolides have been scaled-up in first time pilot plant and kilo-lab campaigns. These building blocks have supported the discovery of new macrolide antibiotics as well as ongoing preclinical studies.

Diastereoselective Michael-Claisen Cyclizations of γ-Oxa-α,ß-unsaturated Ketones en Route to 5-Oxatetracyclines.

5-Oxatetracyclines were synthesized from d-arabinose using sequential Michael-Claisen cyclization reactions via a 5-oxa-AB enone substrate. The 5-oxatetracyclines were found to have poor stability in aqueous buffer (pH 7.4, 37 °C) and showed little to no inhibition of bacterial growth (S. aureus, E. coli).

Anti-proliferative activity of the NPM1 interacting natural product avrainvillamide in acute myeloid leukemia.

Mutated nucleophosmin 1 (NPM1) acts as a proto-oncogene and is present in ~30% of patients with acute myeloid leukemia (AML). Here we examined the in vitro and in vivo anti-leukemic activity of the NPM1 and chromosome region maintenance 1 homolog (CRM1) interacting natural product avrainvillamide (AVA) and a fully syntetic AVA analog. The NPM1-mutated cell line OCI-AML3 and normal karyotype primary AML cells with NPM1 mutations were significantly more sensitive towards AVA than cells expressing wild-type (wt) NPM1. Furthermore, the presence of wt p53 sensitized cells toward AVA. Cells exhibiting fms-like tyrosine kinase 3 (FLT3) internal tandem duplication mutations also displayed a trend toward increased sensitivity to AVA. AVA treatment induced nuclear retention of the NPM1 mutant protein (NPMc+) in OCI-AML3 cells and primary AML cells, caused proteasomal degradation of NPMc+ and the nuclear export factor CRM1 and downregulated wt FLT3 protein. In addition, both AVA and its analog induced differentiation of OCI-AML3 cells together with an increased phagocytotic activity and oxidative burst potential. Finally, the AVA analog displayed anti-proliferative activity against subcutaneous xenografted HCT-116 and OCI-AML3 cells in mice. Our results demonstrate that AVA displays enhanced potency against defined subsets of AML cells, suggesting that therapeutic intervention employing AVA or related compounds may be feasible.

A platform for the discovery of new macrolide antibiotics.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

Development of a platform for the discovery and practical synthesis of new tetracycline antibiotics.

Tetracyclines have proven to be safe and effective antibiotics over decades but to date all approved members of the class have been discovered and manufactured by chemical modification of fermentation products, which greatly limits the number of new structures that can be explored as future medicines. This review summarizes research leading to the development of a platform synthetic technology that enabled the discovery of the clinical candidate eravacycline, as well as other promising new tetracycline antibiotics, and provides the basis for a practical route for their manufacture. The approach argues for a reassessment of other antibiotic classes based on natural products for which practical, fully synthetic routes have not yet been developed, suggesting that these may represent underdeveloped resources with great potential to offer safer and more effective anti-infective agents.