Synthesis and evaluation of troponoids as a new class of antibiotics

Novel antibiotics are urgently needed. The troponoids [tropones, tropolones, and α-hydroxytropolones (α-HT)] can have anti-bacterial activity. We synthesized or purchased 92 troponoids and evaluated their antibacterial activities against Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Preliminary hits were assessed for minimum inhibitory concentrations (MIC80) and cytotoxicity (CC50) against human hepatoma cells. Sixteen troponoids inhibited S. aureus/E. coli/A. baumannii growth by ≥80% growth at 50 values >50 μM. Two selected tropolones (63 and 285) inhibited 18 methicillin-resistant S. aureus (MRSA) strains with similar MIC80 values as against a reference strain. Two selected thiotropolones (284 and 363) inhibited multidrug-resistant (MDR) E. coli with MIC80 ≤30 μM. One α-HT (261) inhibited MDR-A. baumannii with MIC80 ≤30 μM. This study opens new avenues for development of novel troponoid antibiotics to address the critical need to combat MDR bacterial infections

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors

The natural product α-hydroxytropolones manicol and β-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an “RNase H-like” fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein–nucleic acid contacts over a region of ∼14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme–substrate complex to the enzyme–product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe+2). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.Fil: Muzio, Federico Matías. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Masi, Marco. Università degli Studi di Napoli Federico II; ItaliaFil: Tuzi, Angela. Università degli Studi di Napoli Federico II; ItaliaFil: Evidente, Antonio. Università degli Studi di Napoli Federico II; ItaliaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Studies Toward a General Synthesis of Poly-Substituted Alpha-Hydroxytropolones

Chapter 1: This chapter gives a brief history of α-hydroxytropolones, how they were discovered and unique properties of these substrates. Included is a background on the bioactivity of these substrates in cellular targets such as bacteria, fungi, parasites, tumors and toxicity, as well as their ability to inhibit various metallo-based enzymes. Structure activity relationships studies are reviewed on important metalloenzymes HIV-Reverse Transcriptase (RT) and Inositol monophosphatase (IMPase). Finally the chapter finishes with a synthetic overview of α-hydroxytropolones including natural product targets such as puberulic acid, puberulonic acid, β-thujaplicinol, and β-hydroxytropolone. Chapter 2: A brief review on β-hydroxy-γ-pyrone based oxidopyrylium cycloadditions will be presented as well as important oxidopyrylium cycloaddition/ring opening procedures to yield natural tropolone products. Research from the Murelli laboratory will be highlighted. This chapter will discuss a new synthetic route toward functionalized α-hydroxytropolones. A β-hydroxy-γ-pyrone intermolecular oxidopyrylium cycloaddition with a range of alkynes that was optimized to an efficient and high yielding process will be discussed. Next two ring catalyzed ring openings will be discussed; one that utilizes boron trichloride that attains α-hydroxytropolones and 7-methoxytropolones, and a triflic acid mediated sequence that yields exclusive 7-methoyxtropolones and furans. Finally, a new reaction with the oxidopyrylium species will be highlighted that shows the exchange of alcohols in these reactive species. Chapter 3: Chapter three describes the background on three specific medicinal targets: ANT (2 )-Ia, HIV RT RNase H, and HBV RT RNaseH and preliminary structure activity relationship studies with β-hydroxytropolones synthesized in this research are outlined

Metal coordinating inhibitors of Rift Valley fever virus replication

Rift Valley fever virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the order Bunyavirales, whose replication depends on the enzymatic activity of the viral L protein. Screening for RVFV inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 47 novel RVFV inhibitors with selective indexes from 1.1-103 and 50% effective concentrations of 1.2-56 μM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against a second Bunyavirus, La Crosse Virus (LACV), and the flavivirus Zika (ZIKV). These data indicate that the α-Hydroxytropolone and N-Hydroxypyridinedione chemotypes should be investigated in the future to determine their mechanism(s) of action allowing further development as therapeutics for RVFV and LACV, and these chemotypes should be evaluated for activity against related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus

Discovery of Pseudomonas Natural Products Involved in the Biological Control of Potato Pathogens

Potato common scab and late blight, caused by Streptomyces scabies and Phytophthora infestans, respectively, are serious diseases affecting one of the world’s largest and most important food crops. The lack of stable interventions has shifted recent focus toward biological control (biocontrol) methods. Pseudomonas isolates have shown significant promise as bacterial biocontrol agents, occurring in soils worldwide with high inter-strain diversity and potential for natural product biosynthesis. This thesis details investigations into the biosynthetic potential of environmental Pseudomonas strains isolated from a potato field, with a focus on discovering novel natural products active against plant pathogens. Investigations focused on a strain showing strong biocontrol phenotypes, Ps652. Initially, this strain showed strong inhibition of phytopathogens but with few biosynthetic gene clusters (BGCs) identified by common methods. A variety of methods were used to identify the determinants of the strong biocontrol phenotype shown by this strain, including activity-guided isolation of natural products and transposon mutagenesis. 3,7-dihydroxytropolone (3,7-HT) is reported here as being produced by a Pseudomonas isolate for the first time. 3,7-HT shows improved activity towards Streptomyces scabies compared to 7-hydroxytropolone, but does not fully explain activity of Ps652 against P. infestans. Additionally, investigations were made into putative RiPP BGCs containing DUF692 proteins in environmental Pseudomonas isolates Ps706 and Ps708. These BGCs appeared associated with phytopathogen inhibition in previous work, and were studied here using bioinformatics, gene deletions, and heterologous expression approaches

Synthesis and Analysis of Novel Troponoid-Based Chemical Probes

Troponoids are a class of non-benzenoid aromatic species featuring a cycloheptatrienone ring and varying degrees of oxygenation. These scaffolds have proven ubiquitous amongst natural products, and have displayed promise as therapeutic agents against a variety of diseases. Herein, we will describe our efforts towards furthering troponoids as potential pharmaceuticals. In Chapter 1, we outline a kojic acid-derived oxidopyrylium cycloaddition/ring-opening method developed in our lab to generate ahydroxytropolones (aHTs). This route was successfully adapted to synthesize a small library of lipophilic aHTs that were proven to be effective against herpes simplex virus-1 (HSV-1) replication, while providing further insight into the mechanism of troponoid-based HSV-1 inhibition. In Chapter 2, we adapt the oxidopyrylium cycloaddition method to allow for the incorporation of acid-sensitive groups onto the aHT core. We outline our efforts at developing a mild, late-stage direct amidation of aHTs that has enabled access to a range of previously inaccessible functionalities. This amidation methodology was also successfully utilized on tropolone and tropone-containing substrates, proving to be an effective method for amidotroponoid synthesis. v In Chapter 3, we provide a literature review of tropolone syntheses arising from diazo-derived ring expansions. We then describe our discovery and optimization of an ambient air-promoted autoxidation of Buchner ring expansion-derived cycloheptatrienes into aromatic tropolones. The scope and generality of this reaction was assessed, and empirically obtained data supported a robust hypothesis for the putative mechanism of autoxidation. In Chapter 4, a class of amidotroponoids containing a light-reactive diazirine moiety and alkyne reporter tag were assessed for their potential as activity-based probes. The troponoid probes covalently labeled proteins in a live cell line of HEK-293T cells in a UVand concentration-dependent manner, enabling the preliminary identification of novel troponoid protein targets. Additionally, the experiments revealed the ability of troponoids to covalently photolabel proteins in the absence a known photo-labile moiety. The mechanism of this photolabeling, in addition to the characterization of an analogous troponoid-amino acid adduct, is investigated

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 A° . Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator -thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiledmechanistic details of the two-metalion catalysis at atomic resolution

Synthesis of Biologically Active Tropolones and Stereochemically Rich Compounds via Cross-Coupling Reactions

Over the past years the Murelli lab has established research based on the synthesis and biological evaluations of alpha-hydroxytropolones (alpha-HTs). These seven-membered aromatic rings have three contiguous oxygen atoms that chelates to metals, thus, biologically having the ability to bind dinuclear metalloenzymes and promoting inhibition. Synthetic routes to alpha-HT’s are scarce and therefore has been the main driving force in developing efficient and general synthetic methods to obtain variations in alpha-HTs, enabling SAR studies. The Murelli lab have therefore developed an oxidopyrylium cycloaddition/ring-opening strategy that is able to generate polysubstituted alpha-HTs starting from kojic acid, a cheap and readily available reagent. Adding to this, the Murelli lab have developed a scalable and reproducible procedure that obtains oxidopyrylium ylide in high purity without the need for column chromatography. This has enabled the synthesis and biological evaluations of libraries of alpha-HTs for many human diseases including herpes simplex virus, hepatitis B virus and other human pathogens. Atropisomerism is a form of chirality arising from conformational restriction of chiral axes and has an essential role in many compounds including, but not limited to, chiral ligands, and pharmaceutical drugs. Structural aspect of tropolones such as their large ring size, smaller bond angles, tropylium characteristics, potential ring-puckering, and configurational stability have enabled the synthesis of atropisomeric troponoid-benzenoid structures. Our ability to synthesize atropisomeric troponoid-benzenoid compounds, through cross-coupling reactions, paves the way for entry into a whole new ligand class and asymmetric catalysis in contrast to the abundant benzenoid based privileged ligands. The Biscoe lab studies asymmetric C–C bond formation utilizing cross-coupling reactions such as Suzuki and Stille reactions. The Stille reaction is a chemical reaction that involves the use of organotin compounds with a variety of electrophiles. Stereospecific transfer of chirality using configurationally stable enantioenriched carbastannatranes, as the organotin coupling partner, has been realized with control over retention or inversion of stereochemistry. This is accomplished using palladium catalysis in combination with an electron deficient Buchwald type ligand and copper (I) salts. Herein, we describe biological evaluations of synthetic alpha-HTs, identification of conditions for the atropselective cross-coupling of tropolones and benzenoid groups to generate axially chiral tropone-benzenoid biaryl systems. We also describe developments of cross-coupling methods that enables the stereospecific transfer of a stereogenic center utilizing the Stille reaction