Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

Haplo-insufficiency for different genes differentially reduces pathogenicity and virulence in a fungal phytopathogen

The main determinant of pathogenicity in Ustilago maydis is the b-mating locus, where establishment of heterozygosity is sufficient to cause galls/tumors on maize plants. However, matings between haploids where one partner contains a mutation, in e.g., the smu1 gene, encoding a Ste20-like PAK kinase, often show reduced mating and pathogenicity compared to wild type. Here we show that similarly, diploids lacking one copy of smu1, are reduced in production of aerial hyphae, but do not show significantly-reduced virulence. Haplo-insufficiency was also observed for additional genes. UmPde1 is a cyclic phosphodiesterase involved in cAMP turnover as part of the cAMP-dependent PKA pathway. Hsl7 plays a role in cell length and in the filamentous response to low ammonium in haploid cells. Diploids deleted for one copy of either the pde1 or hsl7 genes had reduced or increased production of aerial hyphae, respectively, and both were severely impaired in virulence compared to wild type diploids. rho1 and pdc1 are two genes essential for cell viability in haploids. These genes also displayed haplo-insufficiency for pathogenesis. rho1/Δrho1 diploid cells were defective in pheromone production and detection, aerial hyphae induction, and were avirulent. In contrast, pdc1/Δpdc1 diploid cells only failed to produce tumors when applied to maize whorls. We predict the haplo-insufficiency of most of these signaling components is due to stoichiometric imbalance of the respective gene products with their interacting partners, thereby impairing virulence-induction mechanism(s). Further investigation of the bases for such haplo-insufficiency as well as of additional genes displaying this phenotype will provide important insights into fundamental aspects of development in this organism as well as inter-nuclear communication and genetic control.     

Identification of a Live Attenuated Vaccine Candidate for Tularemia Prophylaxis

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100–1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.     

Photochemical synthesis of substituted indan-1-ones related to donepezil.

A photoenolization reaction is shown to be the key reaction step in the preparation of substituted indan-1-ones as convenient precursors for the synthesis of donepezil, a well-known acetylcholinesterase inhibitor. Model 2,5-dialkylphenacyl chlorides, differently substituted in the alpha-carbon position, were found to produce indan-1-ones upon irradiation in non-nucleophilic solvents in high chemical yields via hydrogen chloride release. While direct excitation of 4,5-dimethoxy-2-methylphenacyl chloride led to a complex mixture of photoproducts, photolysis of the corresponding benzoate was found to form 5,6-dimethoxyindan-1-one in 62-72% chemical yields and a relatively low quantum efficiency (Phi approximately 0.02). This compound can then be easily converted to donepezil by standard synthetic steps described in the literature. Isotopic exchange and quenching experiments revealed that the product is obtained by the photoenolization process via the triplet excited state, while minor side-photoproducts originate from the singlet excited state. Irradiation of the reactant in neat acetone, used both as a triplet sensitizer and solvent at the same time, was found to form 5,6-dimethoxyindan-1-one exclusively in high (90%) chemical yield.     

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos^−/−, and phox^−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.