The Discovery of Weddellamycin, a Tricyclic Polyene Macrolactam Antibiotic from an Antarctic Deep-Sea-Derived Streptomyces sp. DSS69, by Heterologous Expression.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10-0.83 µg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 µg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

New polycyclic tetramate macrolactams with antimycobacterial activity produced by marine-derived Streptomyces sp. KKMA-0239.

During our screening for anti-mycobacterial agents against Mycobacterium avium complex (MAC), two new polycyclic tetramate macrolactams (PTMs), named hydroxycapsimycin (1) and brokamycin (2), were isolated along with the known PTM, ikarugamycin (3), from the culture broth of marine-derived Streptomyces sp. KKMA-0239. The relative structures of 1 and 2 were elucidated by spectroscopic data analyses, including 1D and 2D NMR. Furthermore, the absolute configuration of 1 was confirmed by a single-crystal X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate antimycobacterial activities against MAC, including clinically isolated drug-resistant M. avium.

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non-structural 3/4A (NS3/4A) serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

Structurally Diverse Macrolactams from an Antarctic Streptomyces Species.

Twelve new polyenic macrolactams, cyclamenols G-R (1 and 3-13), together with the known analogue cyclamenol A (2), were isolated from Streptomyces sp. OUCMDZ-4348. Their structures were elucidated by spectroscopic analysis, quantum chemical calculations, chemical derivatizations, and Mosher's methods. The sequenced genome of OUCMDZ-4348 revealed the putative biosynthetic gene cluster of cyclamenols. It was proposed that the polycyclic natural products, cyclamenols H-R, might be formed from cyclamenols A and G through nonenzymatic intramolecular cycloadditions and oxidative cyclizations.

Nagimycins A and B, Antibacterial Ansamycin-Related Macrolactams from Streptomyces sp. NA07423.

Chemical investigation of Streptomyces sp. NA07423 led to the discovery of two unreported macrolactams, nagimycins A (1) and B (2). Their structures were elucidated by NMR, HRESIMS, X-ray crystallography, and comparison of experimental and theoretical ECD spectra. The nagimycins have a unique butenolide moiety rarely found in ansamycin antibiotics. Genome analysis revealed the putative biosynthetic gene cluster for nagimycins, and a likely biosynthetic pathway was proposed. Notably, compounds 1 and 2 exhibited potent antibacterial activity against two pathogenic Xanthomonas bacteria.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Modifications, biological origin and antibacterial activity of naphthalenoid ansamycins.

Covering: 2011 to 2021Structural division of natural naphthalenoid ansamycins, regarding the type of the core and length of the ansa chain, and their biosynthetic pathways in microorganisms are discussed. The great biosynthetic plasticity of natural naphthalenoid ansamycins is reflected in their structural variety due to the alterations within ansa bridge or naphthalenoid core portions. A comparison between the biological potency of natural and semisynthetic naphthalenoid ansamycins was performed and discussed in relation to the molecular targets in cells. The antibacterial potency of naphthalenoid ansamycins seems to be dependent on the ansa chain length and conformational flexibility - the higher flexibility of the ansa chain the better biological outcome is noted.

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance.

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

Human hepatitis B virus-derived virus-like particle as a drug and DNA delivery carrier.

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators.

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1-7) and by quinuclidine motif (8), transformed into ammonium salts (9-13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC50s = 0.09-1.06 µM). Transformation of 8 into salts 9-13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC50∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC50∼2 µM to IC50∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8-13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.

Heat Shock Protein 90 Inhibitor Effects on Pancreatic Cancer Cell Cultures.

OBJECTIVES: Pancreatic ductal adenocarcinoma is one of the deadliest cancers for which few curative therapies are available to date. Heat shock protein 90 (Hsp90) inhibitors have shown activity against numerous cancers in vitro; therefore, we tested whether they could be used to target pancreatic ductal adenocarcinoma. METHODS: Inhibitors of Hsp90 ATPase activity were applied on low-passage pancreatic cell line cultures (Panc10.05, Panc215, A6L) in a dose-response manner, and the inhibitor in vitro effect on cell growth was evaluated. Seven of novel Hsp90 inhibitors based on resorcinol fragment and 5 commercially available Hsp90 inhibitors (17-AAG, AT-13387, AUY-922, ganetespib, and rifabutin) as well as control compound triptolide were tested yielding IC50 values in 2- and 3-dimensional assays. RESULTS: The novel Hsp90 inhibitors exhibited strong effects on all 3 tested pancreatic cell line cultures (Panc10.05, Panc215, A6L) reaching the IC50 of 300 to 600 nM in 2- and 3-dimensional assays. CONCLUSIONS: Novel Hsp90 inhibitors can be developed as antipancreatic cancer agents. Their chemical structures are simpler, and they are likely to exhibit lower side effects than the much more complex inhibitors used as controls.

A Smart Hyperthermia Nanofiber-Platform-Enabled Sustained Release of Doxorubicin and 17AAG for Synergistic Cancer Therapy.

This study demonstrates the rational fabrication of a magnetic composite nanofiber mesh that can achieve mutual synergy of hyperthermia, chemotherapy, and thermo-molecularly targeted therapy for highly potent therapeutic effects. The nanofiber is composed of biodegradable poly(ε-caprolactone) with doxorubicin, magnetic nanoparticles, and 17-allylamino-17-demethoxygeldanamycin. The nanofiber exhibits distinct hyperthermia, owing to the presence of magnetic nanoparticles upon exposure of the mesh to an alternating magnetic field, which causes heat-induced cell killing as well as enhanced chemotherapeutic efficiency of doxorubicin. The effectiveness of hyperthermia is further enhanced through the inhibition of heat shock protein activity after hyperthermia by releasing the inhibitor 17-allylamino-17-demethoxygeldanamycin. These findings represent a smart nanofiber system for potent cancer therapy and may provide a new approach for the development of localized medication delivery.

The Odd Couple(s): An Overview of Beta-Lactam Antibiotics Bearing More Than One Pharmacophoric Group.

ß-lactam antibiotics are among the most important and widely used antimicrobials worldwide and are comprised of a large family of compounds, obtained by chemical modifications of the common scaffolds. Usually these modifications include the addition of active groups, but less frequently, molecules were synthesized in which either two ß-lactam rings were joined to create a single bifunctional compound, or the azetidinone ring was joined to another antibiotic scaffold or another molecule with a different activity, in order to create a molecule bearing two different pharmacophoric functions. In this review, we report some examples of these derivatives, highlighting their biological properties and discussing how this strategy can lead to the development of innovative antibiotics that can represent either novel weapons against the rampant increase of antimicrobial resistance, or molecules with a broader spectrum of action.

Lessons from Natural Product Total Synthesis: Macrocyclization and Postcyclization Strategies.

Macrocyclic natural products are plentiful in the bacteria, archaea, and eukaryote domains of life. For the significant advantages that they provide to the producing organisms, evolution has learned how to implement various types of macrocyclization reactions into the different biosynthetic pathways and how to effect them with remarkable ease. Mankind greatly benefits from nature's pool, not least because naturally occurring macrocycles or derivatives thereof serve as important drugs for the treatment of many serious ailments.In stark contrast, macrocyclization reactions are usually perceived as difficult to accomplish by purely chemical means. While it is true that ring closure necessarily entails an entropic loss and may result in the buildup of (considerable) ring strain that must be compensated for in one way or the other, it is also fair to note tremendous methodological advances during the last decades that greatly alleviated this traditional "macrocycle challenge". It is therefore increasingly possible to explore the advantages provided by large as well as medium-size ring systems in a more systematic manner. This venture also holds the promise of increasing the "chemical space" amenable to drug development to a considerable extent.In consideration of this and other important long-term perspectives, it is appropriate to revisit the current state of the art. To this end, a number of vignettes are presented, each of which summarizes a total synthesis project targeting macrocyclic natural products of greatly different chemotypes using a variety of transformations to reach these goals. Although we were occasionally facing "dead ends", which are also delineated for the sake of a complete picture, these case studies illustrate the notion that the formation of a certain macrocyclic perimeter is (usually) no longer seriously limiting. In addition to substantial progress in the "classical" repertoire (macrolactonization and macrolactamization (pateamine A, spirastrellolide, and belizentrin)), various metal-catalyzed reactions have arguably led to the greatest leaps forward. Among them, palladium-catalyzed C-C bond formation (roseophilin and nominal xestocyclamine A) and, in particular, alkene and alkyne metathesis stand out (iejimalide, spirastrellolide, enigmazole, ingenamine, and sinulariadiolide). In some cases, different methods were pursued in parallel, thus allowing for a critical assessment and comparison.To the extent that the macrocyclic challenge is vanishing, the opportunity arises to focus attention on the postmacrocyclization phase. One may stipulate that a well-designed cyclization precursor does not only ensure efficient ring closure but also fosters and streamlines the steps that come after the event. One way to do so is dual (multiple) use in that the functional groups serving the actual cyclization reaction also find productive applications downstream from it rather than being subject to simple defunctionalization. In this context, better insight into the conformational peculiarities of large rings and the growing confidence in their accessibility in a stereochemically well defined format rejuvenate the implementation of transannular reactions or reaction cascades that can lead to rapid and substantial increases in molecular complexity. The examples summarized herein showcase such possibilities, with special emphasis on tranannular gold catalysis and the emerging ruthenium-catalyzed trans-hydrometalation chemistry for the selective functionalization of alkynes.

Design, Synthesis and In Vitro Investigation of Novel Basic Celastrol Carboxamides as Bio-Inspired Leishmanicidal Agents Endowed with Inhibitory Activity against Leishmania Hsp90.

The natural triterpene celastrol (CE) is here used as lead compound for the design and synthesis of a panel of eleven CE carboxamides that were tested in vitro for their growth inhibitory activity against Leishmania infantum and L.tropica parasites. Among them, in vitro screening identified four basic CE carboxamides endowed with nanomolar leishmanicidal activity, against both the promastigotes and the intramacrophage Leishmania amastigotes forms. These compounds also showed low toxicity toward two human (HMEC-1 and THP-1) and one murine (BMDM) cell lines. Interestingly, the most selective CE analogue (compound 3) was also endowed with the ability to inhibit the ATPase activity of the Leishmania protein chaperone Hsp90 as demonstrated by the in vitro assay conducted on a purified, full-length recombinant protein. Preliminary investigations by comparing it with the naturally occurring Hsp90 active site inhibitor Geldanamycin (GA) in two different in vitro experiments were performed. These promising results set the basis for a future biochemical investigation of the mode of interaction of celastrol and CE-inspired compounds with Leishmania Hsp90.

Molecular Docking and Virtual Screening Based Prediction of Drugs for COVID-19.

AIMS: To predict potential drugs for COVID-19 by using molecular docking for virtual screening of drugs approved for other clinical applications. BACKGROUND: SARS-CoV-2 is the betacoronavirus responsible for the COVID-19 pandemic. It was listed as a potential global health threat by the WHO due to high mortality, high basic reproduction number, and lack of clinically approved drugs and vaccines. The genome of the virus responsible for COVID-19 has been sequenced. In addition, the three-dimensional structure of the main protease has been determined experimentally. OBJECTIVE: To identify potential drugs that can be repurposed for treatment of COVID-19 by using molecular docking based virtual screening of all approved drugs. METHODS: A list of drugs approved for clinical use was obtained from the SuperDRUG2 database. The structure of the target in the apo form, as well as structures of several target-ligand complexes, were obtained from RCSB PDB. The structure of SARS-CoV-2 Mpro determined from X-ray diffraction data was used as the target. Data regarding drugs in clinical trials for COVID-19 was obtained from clinicaltrials.org. Input for molecular docking based virtual screening was prepared by using Obabel and customized python, bash, and awk scripts. Molecular docking calculations were carried out with Vina and SMINA, and the docked conformations were analyzed and visualized with PLIP, Pymol, and Rasmol. RESULTS: Among the drugs that are being tested in clinical trials for COVID-19, Danoprevir and Darunavir were predicted to have the highest binding affinity for the Main protease (Mpro) target of SARS-CoV-2. Saquinavir and Beclabuvir were identified as the best novel candidates for COVID-19 therapy by using Virtual Screening of drugs approved for other clinical indications. CONCLUSION: Protease inhibitors approved for treatment of other viral diseases have the potential to be repurposed for treatment of COVID-19.

Are Computational Methods Useful for Structure Elucidation of Large and Flexible Molecules? Belizentrin as a Case Study.

Quantum mechanical NMR methods are progressively becoming decisive in structure elucidation. However, problems arise using low-level calculations for complex molecules, whereas methods using higher levels of theory are not practical for large molecules. This report outlines a synergistic effort employing computationally inexpensive quantum mechanical NMR calculations with conformer selection incorporating 3JHH values as a way to solve the structure of large, complex, and highly flexible molecules using readily available computational resources with belizentrin as a case study.

The HSP90 Inhibitor, 17-AAG, Influences the Activation and Proliferation of T Lymphocytes via AKT/GSK3ß Signaling in MRL/lpr Mice.

OBJECTIVE: To explore the molecular mechanism of 17-AAG in the treatment of systemic lupus erythematosus (SLE), and the effects of the heat shock protein 90 (HSP90) inhibitor 17-AAG on the activation and proliferation of lymphocytes and the AKT/GSK3ß signaling pathway in MRL/lpr mice were detected. METHODS: MRL/lpr mice were randomly divided into the control group and the experimental group. The experimental group was injected intraperitoneally with 17-AAG, and T lymphocytes were separated by magnetic beads. Lymphocyte proliferation was detected by MTT and flow cytometry (FCM), and the expression of the HSP90 protein and PI3K/AKT signaling pathway-related proteins was detected by Western blotting. Renal histopathology and immune complex deposition were also observed in both groups. RESULTS: Immune complex deposition and inflammation decreased in kidneys from MRL/lpr mice in the experimental group. HSP90 protein expression, T lymphocyte proliferation and phosphorylated AKT and GSK3ß levels also decreased in the experimental group. CONCLUSION: 17-AAG can inhibit the activation and proliferation of T lymphocytes and downregulate the AKT/GSK3ß signaling pathway, which may be relevant for the treatment of SLE.

Geldanamycin-Derived HSP90 Inhibitors Are Synthetic Lethal with NRF2.

Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.

Danoprevir for the Treatment of Hepatitis C Virus Infection: Design, Development, and Place in Therapy.

On June 8, 2018, an NS3/4A protease inhibitor called danoprevir was approved in China to treat the infections of HCV genotype (GT) 1b - the most common HCV genotype worldwide. Based on phase 2 and 3 clinical trials, the 12-week regimen of ritonavir-boosted danoprevir (danoprevir/r) plus peginterferon alpha-2a and ribavirin offered 97.1% (200/206) of sustained virologic response at post-treatment week 12 (SVR12) in treatment-naïve non-cirrhotic patients infected with HCV genotype 1b. Adverse events such as anemia, fatigue, fever, and headache were associated with the inclusion of peginterferon alpha-2a and ribavirin in the danoprevir-based regimen. Moreover, drug resistance to danoprevir could be traced to amino acid substitutions (Q80K/R, R155K, D168A/E/H/N/T/V) near the drug-binding pocket of HCV NS3 protease. Despite its approval, the clinical use of danoprevir is currently limited to its combination with peginterferon alpha-2a and ribavirin, thereby driving its development towards interferon-free, ribavirin-free regimens with improved tolerability and adherence. In the foreseeable future, pan-genotypic direct-acting antivirals with better clinical efficacy and less adverse events will be available to treat HCV infections worldwide.