Novel antibody-antibiotic conjugate using KRM-1657 as payload eliminates intracellular MRSA in vitro and in vivo.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S.aureus within host cells may cause long-term colonization and clinical failure. Current treatments have poor efficacy in clearing intracellular bacteria. Antibody-antibiotic conjugates (AACs) is a novel strategy for eliminating intracellular bacteria. Herein, we use KRM-1657 as payload of AAC for the first time, and we conjugate it with anti S. aureus antibody via a dipeptide linker (Valine-Alanine) to obtain a novel AAC (ASAK-22). The ASAK-22 exhibits good in vitro pharmacokinetic properties and inhibitory activity against intracellular MRSA, with 100 µg/mL of ASAK-22 capable of eliminating intracellular MRSA to the detection limit. Furthermore, the in vivo results demonstrate that a single administration of ASAK-22 significantly reduces the bacterial burden in the bacteremia model, which is superior to the vancomycin treatment.

Genome-Driven Discovery of Hygrocins in Streptomyces rapamycinicus.

Ansamycins, represented by the antituberculosis drug rifamycin, are an important family of natural products. To obtain new ansamycins, Streptomyces rapamycinicus IMET 43975 harboring an ansamycin biosynthetic gene cluster was fermented in a 50 L scale, and subsequent purification work led to the isolation of five known and four new analogues, where hygrocin W (2) belongs to benzoquinonoid ansamycins, and the other three hygrocins, hygrocins X-Z (6-8), are new seco-hygrocins. The structures of ansamycins (1-8) were determined by the analysis of spectroscopic (1D/2D NMR and ECD) and MS spectrometric data. The Baeyer-Villiger enzyme which catalyzed the ester formation in the ansa-ring was confirmed through in vivo CRISPR base editing. The discovery of these compounds further enriches the structural diversity of ansamycins.

Targeted delivery of rifaximin using P6.2-decorated bifunctional PLGA nanoparticles for combating Staphylococcus aureus infections.

The emergence of antibiotic resistance makes the treatment of bacterial infections difficult and necessitates the development of alternative strategies. Targeted drug delivery systems are attracting great interest in overcoming the limitations of traditional antibiotics. Here, we aimed for targeted delivery of rifaximin (RFX) by decorating RFX-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) with synthetic P6.2 peptide, which was used as a targeting agent for the first time. Our results showed that encapsulation of RFX into NPs increased its antibacterial activity by improving its solubility and providing controlled release, while P6.2 modification allowed targeting of NPs to S. aureus bacterial cells. A promising therapeutic approach for bacterial infections, these P6.2-conjugated RFX-loaded PLGA NPs (TR-NP) demonstrated potent antibacterial activity against both strains of S. aureus. The antibacterial activity of RFX-loaded PLGA NPs (R-NP) showed significant results with an increase of 8 and 16-fold compared to free RFX against S. aureus and MRSA, respectively. Moreover, the activity of targeted nanoparticles was found to be increased 32 or 16-fold with an MBC value of 0.0078 µg/mL. All nanoparticles were found to be biocompatible at doses where they showed antimicrobial activity. Finally, it revealed that P6.2-conjugated targeted nanoparticles extremely accumulated in S. aureus rather than E. coli.

Redesign of Rifamycin Antibiotics to Overcome ADP-Ribosylation-Mediated Resistance.

Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity. Validated by a combination of biochemical, structural, and microbiological studies, the most potent analogs overcome ADP-ribosylation, restored their intrinsic low nanomolar activity and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning drug disposition properties afforded a preclinical candidate with remarkable potency and an outstanding pharmacokinetic profile.

Novel C-3-(N-alkyl-aryl)-aminomethyl rifamycin SV derivatives exhibit activity against rifampicin-resistant Mycobacterium tuberculosis RpoBS522L strain and display a different binding mode at the RNAP ß-subunit site compared to rifampicin.

Antimicrobial resistance is a main concern in tuberculosis treatment and is often associated with the emergence of Mycobacterium tuberculosis strains resistant to rifampicin (RIF), which is one of the cornerstones of tuberculosis chemotherapy. In this study, aminoalkyl-aromatic ring tails were appended to the C3 position of rifamycin core to assess the role of C3 substitutions to the anti-mycobacterial activity of the rifamycin antibiotics. The typical hydrazone unit of RIF was replaced by an amino-alkyl linkage to connect the aromatic ring tails with the rifamycin naphthoquinone core. Eight novel C3-(N-alkyl-aryl)-aminoalkyl analogues of rifamycin SV were synthesised and screened in vitro against wild-type HR37Rv and "hypervirulent" HN-878 strains, and a panel of rifampicin-resistant M. tuberculosis clinical isolates carrying mutations at the 522, 531 and 455 positions of the rpoB gene (RpoBS522L, RpoBS531L and RpoBH455D strains). The analogues exhibited anti-tubercular activity against H37Rv and HN-878 at submicromolar or nanomolar concentrations, and against clinical H37Rv isolates bearing the S522L mutations at low micromolar concentration. Benzylamine moiety-including analogue 8 was as active as rifampicin against HN-878 with a MIC90 value of 0.02 µM, whereas 14 and 15, which included tryptamine and para-methyl-sulfonylbenzylamine C3-substituents, respectively, showed higher anti-tubercular activity (MIC90 = 3 µM) compared to rifampicin against the S522L mutated H37Rv strain. Detailed in silico analysis of different RNAP molecular systems predicted a distinct, possibly novel, binding mode for the new rifamycin analogues. These were found to occupy a different space in the binding pockets of both wild type and mutated RNAP proteins compared to that of rifampicin. Moreover, the molecular modelling experiments investigated the ability of the novel analogues aromatic tails to establish key interactions at the RNAP binding site. These interesting findings might pave the way for generating rifamycin analogues that can overcome anti-microbial resistance in M. tuberculosis.

Rifamycin W Analogues from Amycolatopsis mediterranei S699 Δrif-orf5 Strain.

Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1-13) including seven new ones (1-7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1-4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1-3, 9 and 10 featured a hemiacetal. Compounds 5-7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1-3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.

The Enzymes of the Rifamycin Antibiotic Resistome.

Rifamycin antibiotics include the WHO essential medicines rifampin, rifabutin, and rifapentine. These are semisynthetic derivatives of the natural product rifamycins, originally isolated from the soil bacterium Amycolatopsis rifamycinica. These antibiotics are primarily used to treat mycobacterial infections, including tuberculosis. Rifamycins act by binding to the ß-subunit of bacterial RNA polymerase, inhibiting transcription, which results in cell death. These antibiotics consist of a naphthalene core spanned by a polyketide ansa bridge. This structure presents a unique 3D configuration that engages RNA polymerase through a series of hydrogen bonds between hydroxyl groups linked to the naphthalene core and C21 and C23 of the ansa bridge. This binding occurs not in the enzyme active site where template-directed RNA synthesis occurs but instead in the RNA exit tunnel, thereby blocking productive formation of full-length RNA. In their clinical use to treat tuberculosis, resistance to rifamycin antibiotics arises principally from point mutations in RNA polymerase that decrease the antibiotic's affinity for the binding site in the RNA exit tunnel. In contrast, the rifamycin resistome of environmental mycobacteria and actinomycetes is much richer and diverse. In these organisms, rifamycin resistance includes many different enzymatic mechanisms that modify and alter the antibiotic directly, thereby inactivating it. These enzymes include ADP ribosyltransferases, glycosyltransferases, phosphotransferases, and monooxygenases.ADP ribosyltransferases catalyze group transfer of ADP ribose from the cofactor NAD+, which is more commonly deployed for metabolic redox reactions. ADP ribose is transferred to the hydroxyl linked to C23 of the antibiotic, thereby sterically blocking productive interaction with RNA polymerase. Like ADP ribosyltransferases, rifamycin glycosyl transferases also modify the hydroxyl of position C23 of rifamycins, transferring a glucose moiety from the donor molecule UDP-glucose. Unlike other antibiotic resistance kinases that transfer the γ-phosphate of ATP to inactivate antibiotics such as aminoglycosides or macrolides, rifamycin phosphotransferases are ATP-dependent dikinases. These enzymes transfer the ß-phosphate of ATP to the C21 hydroxyl of the rifamycin ansa bridge. The result is modification of a critical RNA polymerase binding group that blocks productive complex formation. On the other hand, rifamycin monooxygenases are FAD-dependent enzymes that hydroxylate the naphthoquinone core. The result of this modification is untethering of the ansa chain from the naphthyl moiety, disrupting the essential 3D shape necessary for productive RNA polymerase binding and inhibition that leads to cell death.All of these enzymes have homologues in bacterial metabolism that either are their direct precursors or share common ancestors to the resistance enzyme. The diversity of these resistance mechanisms, often redundant in individual bacterial isolates, speaks to the importance of protecting RNA polymerase from these compounds and validates this enzyme as a critical antibiotic target.

A Flavin-Dependent Monooxygenase Mediates Divergent Oxidation of Rifamycin.

Rifamycins have been clinically utilized against mycobacterial infections for more than 50 years; however, their biosynthesis has not been fully elucidated. Here, on the basis of in vivo gene deletions, in vitro enzyme assays, isotope labeling, and site-directed mutations, we found that a flavin-dependent monooxygenase encoded by a rifamycin biosynthetic gene cluster, Rif-Orf17, not only converted the naphthoquinone chromophore of rifamycin S into benzo-γ-pyrone but also linearized rifamycin SV through phenolic hydroxylation. Both oxidation routes lead to inactivation of rifamycins.

Computational and In Vitro Investigation of (-)-Epicatechin and Proanthocyanidin B2 as Inhibitors of Human Matrix Metalloproteinase 1.

Matrix metalloproteinases 1 (MMP-1) energetically triggers the enzymatic proteolysis of extracellular matrix collagenase (ECM), resulting in progressive skin aging. Natural flavonoids are well known for their antioxidant properties and have been evaluated for inhibition of matrix metalloproteins in human. Recently, (-)-epicatechin and proanthocyanidin B2 were reported as essential flavanols from various natural reservoirs as potential anti-inflammatory and free radical scavengers. However, their molecular interactions and inhibitory potential against MMP-1 are not yet well studied. In this study, sequential absorption, distribution, metabolism, and excretion (ADME) profiling, quantum mechanics calculations, and molecular docking simulations by extra precision Glide protocol predicted the drug-likeness of (-)-epicatechin (-7.862 kcal/mol) and proanthocyanidin B2 (-8.145 kcal/mol) with the least reactivity and substantial binding affinity in the catalytic pocket of human MMP-1 by comparison to reference bioactive compound epigallocatechin gallate (-6.488 kcal/mol). These flavanols in docked complexes with MMP-1 were further studied by 500 ns molecular dynamics simulations that revealed substantial stability and intermolecular interactions, viz. hydrogen and ionic interactions, with essential residues, i.e., His218, Glu219, His222, and His228, in the active pocket of MMP-1. In addition, binding free energy calculations using the Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method suggested the significant role of Coulomb interactions and van der Waals forces in the stability of respective docked MMP-1-flavonol complexes by comparison to MMP-1-epigallocatechin gallate; these observations were further supported by MMP-1 inhibition assay using zymography. Altogether with computational and MMP-1-zymography results, our findings support (-)-epicatechin as a comparatively strong inhibitor of human MMP-1 with considerable drug-likeness against proanthocyanidin B2 in reference to epigallocatechin gallate.

8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain.

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1-11) and seven known analogues (12-18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4-11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 µg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 µM, respectively.

Rifamycin O, An Alternative Anti-Mycobacterium abscessus Agent.

Mycobacterium abscessus is the most difficult-to-treat nontuberculous mycobacteria because of its resistance to many antibiotics. In this study, we screened the Korea Chemical Bank library for a bioluminescent reporter assay to identify molecules capable of acting against M. abscessus. On application of the assay, rifamycin O showed excellent in vitro activity with a narrow range of the minimum inhibitory concentration required to inhibit the growth of 90% of the bacterium (MIC90 = 4.0-6.2 µM); its in vivo efficacy in the zebrafish (Danio rerio) infection model was comparable to that of rifabutin at 25 µM. Furthermore, rifamycin O did not show significant toxicity in cells and the zebrafish model. These results are the first in vivo indication that rifamycin O may be a drug candidate for treating M. abscessus infections.

Specific Interactions between Rifamycin Antibiotics and Water Influencing Ability To Overcome Natural Cell Barriers and the Range of Antibacterial Potency.

Rifamycins are a group of macrocyclic antibiotics mainly used for the treatment of various bacterial infections including tuberculosis. Spectroscopic studies of rifamycins evidence the formation of temperature- and solvent-dependent equilibria between A-, B-, and C-type conformers in solutions. The B- and C-type conformers of rifamycin antibiotics are exclusively formed in the presence of water molecules. A- and B-type conformers exhibit a hydrophilic and "open" ansa-bridge nature whereas the C-type conformer is more lipophilic due to the presence of a "closed" ansa-bridge structure. The involvement of the lactam moiety of the ansa-bridge in intramolecular H-bonds within rifapentine and rifampicin implicates the formation of a more hydrophilic A-type conformer. In contrast to rifampicin and rifapentine, for rifabutin and rifaximin, the "free" lactam group enhances conformational flexibility of the ansa-bridge, thereby enabling interconversion between A- and C-type conformers. In turn, an equilibrium between A- and C-type conformers for rifamycins improves their adaptation to the changing nature of bacteria cell membranes, especially those of Gram-negative strains and/or to efflux pump systems.

Synthesis, docking and antibacterial studies of more potent amine and hydrazone rifamycin congeners than rifampicin.

New rifamycin congeners (1-33) with incorporated amine and hydrazone substituents leading to lipophilic and/or basic nature and altered rigidity of modified C(3) arm were synthesized and structurally characterized in detail. NMR spectroscopic studies at different temperatures indicate two types of structures of rifamycin congeners that are realized in solution: zwitterionic and non-ionic forms in dependence of the basicity of modified C(3) arm. The presence of rifamycin congeners in these two possible forms has a significant impact on the physico-chemical parameters such as lipophilicity (clogP) and water solubility and different binding mode of the C(3) arm of antibiotic at RNAP binding pocket (molecular target) leading to different antibacterial potency. The highest antibacterial potency against S. aureus (including MRSA and MLSB strains) and S. epidermidis strains, even higher than reference rifampicin (Rif) and rifaximin (Rifx) antibiotics, was found for rifamycin congeners bearing at the C(3) arm relatively rigid and basic substituents (bipiperidine and guanidine groups). These modifications provide favorable docking mode and excellent water solubility resulting in high potency (MICs 0.0078 µg/mL what gives ∼ 8.5 nM), irrespective whether rifamycin congener is a tertiary amine (15) or hydrazone (29). In turn, for a higher antibacterial potency of rifamycin congeners against E. faecalis strain (MICs 0.5 µg/mL that is 0.6 µM) as compared to Rif and Rifx, the most crucial factors are: bulkiness and the lipophilic character of the end of the C(3) rebuilt arm.

Isolation of 11,12- seco-Rifamycin W Derivatives Reveals a Cleavage Pattern of the Rifamycin Ansa Chain.

This study reported the isolation and characterization of 11 rifamycin congeners including six new ones (1-6) from the agar fermentation extract of Amycolatopsis mediterranei S699. Compounds 1 and 2 are rifamycin glycosides named as rifamycinosides A and B, respectively. Their polyketide skeleton represents a novel cleavage pattern of the rifamycin ansa chain. Compounds 6 and 8 showed potential T3SS inhibitory activity, and 6 induced G2/M phase arrest and caused DNA damage in HCT116 cells.

Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase.

Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antibacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching bacterial genome and metagenome sequences for sequences similar to pumJ, the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Effect of the alkyl group in the piperazine N-substitution on the therapeutic action of rifamycins: A drug-membrane interaction study.

In this work, we studied the effects of the N-alkyl group (methyl, cyclopentyl) in the piperazine ring of, respectively, rifampicin (RIF) and rifapentine (RPT) to correlate this substitution with their differential pharmacokinetic properties and overall clinical performance. Since this group is their only structural change, and given that they share the same pharmacological target, differences in their therapeutic behavior may respond to this asset, particularly in their interaction with lipid membranes across the organism. In this study, surface pressure-area isotherms, as well as spectroscopic and microscopic techniques of characterization of phospholipid monolayers at the air/water interface were used to gain insight into drug-membrane interactions. Differences in the affinity for lipid membranes for both drugs, given by the vibration frequency of characteristic chemical groups in the lipid, as well as by reflectivity and mean molecular area of the monolayer, seem to be due to the N-alkyl substituent and can contribute to provide a molecular explanation as why they pose different choices in the chemotherapy against the deadliest infectious disease, tuberculosis.

Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action.

Rifamycin monooxygenases (Rox) are present in a variety of environmental bacteria and are associated with decomposition of the clinically utilized antibiotic rifampin. Here we report the structure and function of a drug-inducible rox gene from Streptomyces venezuelae, which encodes a class A flavoprotein monooxygenase that inactivates a broad range of rifamycin antibiotics. Our findings describe a mechanism of rifamycin inactivation initiated by monooxygenation of the 2-position of the naphthyl group, which subsequently results in ring opening and linearization of the antibiotic. The result is an antibiotic that no longer adopts the basket-like structure essential for binding to the RNA exit tunnel of the target RpoB, thereby providing the molecular logic of resistance. This unique mechanism of enzymatic inactivation underpins the broad spectrum of rifamycin resistance mediated by Rox enzymes and presents a new antibiotic resistance mechanism not yet seen in microbial antibiotic detoxification.

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A, a new enediyne natural product from Streptomyces sp. CB03234.

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase ß-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L-1 in shaking flasks, and 13 ± 1 mg L-1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L-1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.