A Chemoproteomic Approach to Elucidate the Mechanism of Action of 6-Azasteroids with Unique Activity in Mycobacteria.

By illuminating key 6-azasteroid-protein interactions in both Mycobacterium tuberculosis (Mtb) and the closely related model organism Mycobacterium marinum (Mm), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously (ACS Infect. Dis. 2019, 5 (7), 1239-1251) to study their phenotypic effects on Mtb and Mm, both alone and in combination with bedaquiline. The 6-azasteroid analog, 17ß-[N-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in Mm bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 µM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3ß-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of Mtb to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid-protein interactions in Mm whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential Mm/Mtb protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.

Inhibition of Phytosterol Biosynthesis by Azasterols.

Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.

Discovery and development of azasteroids as anticancer agents.

Cancer is the second leading cause of death worldwide following cardiovascular diseases. Cancer can be treated by a variety of techniques including surgery, radiation therapy, immunotherapy, and chemotherapy. Choice of the method can be made based on type, physiologic location and the stage of disease progression. Among chemical methods, steroids find broad applications. Azasteroids have N- substitutions in steroidal rings. This structural modification renders azasteroids advantageous in increased effectiveness and reduced side effects. Numerous accounts of cancer efficacy of this family of compounds are available in literature. The progress made in the discovery, synthetic efforts and development of azasteroids as anticancer agents is broadly outlined in this review.

Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities.

One-third of the world's population carries Mycobacterium tuberculosis (Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 µM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.

Synthesis of D-Ring Annulated Pyridosteroids from ß-Formyl Enamides and Their Biological Evaluations.

Herein, we report the synthesis of a novel class of substituted androst[17,16- b]pyridines (pyridosteroids) from the reaction of ß-formyl enamides with alkynes in high yields. The optimized reaction protocol was extended to acyclic and cyclic ß-formyl enamides to afford nonsteroidal pyridines. Cell survival assay of all compounds were carried against prostate cancer PC-3 cells wherein 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine showed the highest cytotoxic activity. Phase contrast microscopy and flow cytometry studies exhibited marked morphological features characteristic of apoptosis in 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine and abiraterone treated PC-3 cells. The treatment of 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine induces G2/M phase cell cycle arrest in prostate cancer PC-3 cells. Enhancement of apoptotic inductions of PC-3 cells by 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine and abiraterone through the activation of caspases-6, -7, and -8 pathways were supported by qRT-PCR. In silico study of the compound 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine showed stable and promising interaction with the key caspase proteins. Our studies revealed that the pyridosteroid 3-hydroxy-5-en-2',3'-dicarbethoxy-androst[17,16- b]pyridine, bearing pyridine-2,3-dicarbethoxy pharmacophore, facilitated initiation of caspase-8 and activates downstream effectors caspase-6 and caspase-7 and thereby triggering apoptosis of PC-3 cancer cells.

Proteoglycan 4 and hyaluronan as boundary lubricants for model contact lens hydrogels.

Clinical data show that in vitro contact lens friction is related to in vivo comfort. Solutions of biological lubricants hyaluronan (HA) and proteoglycan 4 (PRG4, also known as lubricin) reduce friction at a cornea-polydimethylsiloxane (PDMS) interface. The purpose of this study was to (1) determine if PRG4 can sorb to and lubricate model contact lens materials and (2) assess the boundary lubricating ability of PRG4 and HA compared to saline on model contact lens materials. PRG4 was obtained from bovine cartilage culture and suspended in saline at 300 µg/mL. N,N-Dimethylacrylamidetris (trimethylsiloxy) silane, (DMAA/TRIS) and methacryloxypropyltris (trimethylsiloxy) silane (pHEMA/TRIS) silicone hydrogels were prepared. A previously described in vitro eyelid-hydrogel and cornea-hydrogel biomechanical friction test was used to determine boundary lubricant effect. PRG4 sorption to the hydrogels was assessed using a soak-rinse protocol and western blotting. PRG4 effectively lubricated both silicone hydrogel materials and HA effectively lubricated pHEMA/TRIS, as indicated by a statistically significant reduction in friction compared to the saline control lubricant. An HA and PRG4 combination showed a synergistic effect for pHEMA/TRIS and effectively lubricated DMAA/TRIS. Biological boundary lubricants HA and PRG4 were shown to effectively lubricate silicone hydrogels when in solution. Additionally, HA and PRG4 showed synergistic lubrication for pHEMA/TRIS. The purpose of this study was not to replicate the friction coefficients of contact lenses, but rather to investigate lubricant-surface interactions for common contact lens constituents. These findings contribute to the potential development of biomolecule based lubricant drops for contact lens wearers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1329-1338, 2018.

Targeting progesterone metabolism in breast cancer with l-proline derived new 14-azasteroids.

Breast cancer cell proliferation is promoted by a variety of mitogenic signals. Classically estrogen is considered as most predominant mitogenic signal in hormone-dependent breast cancer and progesterone is primarily considered to have protective effect. However, it is suggested that some progesterone metabolite may promote breast cancer and progesterone metabolites like 5α-pregnane and 4-pregnene could serve as regulators of estrogen-responsiveness of breast cancer cells. Here, we estimated the potential of alternate targeting of breast cancer via progesterone signalling. l-Proline derived novel 14-azasteroid compounds were screened against MCF-7 and MDA-MB-231 cell lines using MTT assay. In silico studies, cell cycle, Annexin-V-FITC/PI, JC-1 mitochondrial assay, ROS analysis were performed to analyse the impact of hit compound 3b on breast cancer cells. Further, we analysed the impact of hit 3b on the progesterone, its metabolites and enzymes responsible for the conversion of progesterone and its metabolites using ELISA. Data suggests that compound 3b binds and down regulates of 5α-reductase by specifically inhibiting production of progesterone metabolites that are capable of promoting breast cancer proliferation, epithelial mesenchymal transition and migration. This study establishes the proof of concept and generation of new leads for additional targeting of breast cancer.

Generation of comparative pharmacophoric model for steroidal 5α-reductase I and II inhibitors: A 3D-QSAR study on 6-azasteroids.

Steroidal 5α-reductase, a key enzyme involved in the transformation of testosterone to dihydrotestosterone, is unstable during the purification leading to loss of the activity. Therefore, due to unstable nature, the crystal structure of the 5α-reductase is unknown. In the present study, we have generated a comparative pharmacophoric model for both isoforms of steroidal 5α-reductase using 6-azasteroids. The steric and electrostatic maps generated for both isoforms provides structure framework for designing of new inhibitors. Further, 3D-maps are also helpful in understanding variability in the activity of the compounds. Statistical measures generated for both enzymes showed good internal and external prediction. Overall, the analyses of models provides structural requirement of dual and selective steroidal 5α-reductase inhibitors in an interactive fashion.

Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens.

Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

Synthesis and biological evaluation of novel C6-amino substituted 4-azasteroidal purine nucleoside analogues.

Novel C6-amino substituted purine nucleoside analogues (2-12) bearing a modified pyranose-like D ring of the 4-azasteroid moiety were efficiently synthesized through nucleophilic substitution at C6 position of the steroidal nucleoside precursors (1a, b) with versatile amines. All the synthesized new compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 4b, 7b and 9b exhibited significant cytotoxicity with the IC50 values of 2.99 µM (PC-3), 2.84 µM, (PC-3) and 2.69 µM (Hela), respectively.

Synthesis and cytotoxic activity of some 4,6-diaza-A,B-dihomo-steroid bilactams.

Using cholesterol, stigmasterol and sitosterol as starting materials, some 4,6-diaza-A,B-dihomo-steroid bilactams were synthesized via two different synthetic routes by oxidation, reduction, oximation, Beckman rearrangement, etc. The cytotoxic activity of the synthesized compounds against SGC 7901 (human ventriculi carcinoma), Bel-7404 (human liver carcinoma), HeLa (human cervical carcinoma) and HT-29 (colonic carcinoma) cancer cells were investigated. The results showed that compounds 2 and 7b displayed a good cytotoxic activity to the SGC 7901, Bel 7404 and HeLa tumor cell lines with the IC50 values of 11.6, 16.4, 13.9 and 13.1, 21.8, 13.1 µmol/L, respectively. Their cytotoxic activity is almost same as cisplatin to these cells. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Enhanced oral bioavailability and controlled release of dutasteride by a novel dry elixir.

To develop a solid dosage form of dutasteride for improving its oral bioavailability, a novel dry elixir (DE) system was fabricated. DEs incorporating dextrin and/or xanthan gum were prepared using spray-drying and evaluated by morphology, ethanol content, crystallinity, dissolution and oral bioavailability. DEs were spherical with a smooth surface and had an average particle size of 20-25 µm. The ethanol content could be easily varied by controlling the spray-drying temperature. The dissolution profiles of dutasteride from each DE proved to be much faster than that of dutasteride powder due to the amorphous state and a high amount of incorporated ethanol. In particular, the pharmacokinetic profiles of dutasteride were significantly altered depending on the proportions of dextrin and xanthan gum. Blood concentrations of dutasteride from DE formulations were similar to those of market products and much greater than those of native dutasteride. Interestingly, the dissolution and pharmacokinetic profiles were easily controlled by changing the ratio of dextrin to xanthan gum. The data suggests that a DE using dextrin and/or xanthan gum could provide an applicable solid dosage form to improve the dissolution and bio-availability of dutasteride as well as to modulate its pharmacokinetics.

Protein-binding properties of a designed steroidal lactam compound.

Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3ß-hydroxy-17-aza-d-homo-5-androsten-17-one (HAAO), was synthesized and identified. The interactions between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and molecular modeling procedures. It was found that HAAO locates in Sudlow's site I in subdomain IIA of HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO-HSA complexes at ground state. The number of binding sites, binding constants, enthalpy change (ΔH(θ)), Gibbs free energy change (ΔG(θ)) and entropy change (ΔS(θ)) were calculated at different temperatures based on fluorescence quenching theory and classical thermodynamic equation. The percentages content of the HSA's secondary structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with those in no presence of HAAO. In addition, the experimental results of both binding site and conformational change were further confirmed by molecular modeling investigation, in which more details of the binding were visually unfolded. The information provided by the study may be useful for designing novel chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.

Investigation of polymeric excipients for dutasteride solid dispersion and its physicochemical characterization.

To investigate the effects of polymeric excipients for dutasteride solid dispersion, experimental approaches together with physical interactions at molecular level were evaluated. The drug and various polymers (anionic, amphiphilic, and hydrophilic) were mixed physically into different ratios and their thermodynamic and physical properties were analyzed by differential scanning calorimetry and Fourier transform-infrared spectroscopy, respectively. The enhanced equilibrium solubility of dutasteride was also investigated. Dutasteride is non-ionic and showed low solubility in the tested pH ranges (lower than the detection limit of 20 ng/mL). Kollidon(®) MAE 100P, an anionic polymer, showed enhanced dutasteride solubility in aqueous solution followed by hydrophilic Kollidon(®) SR and the amphiphilic polymer, Soluplus(®). Melting point (T m ) of dutasteride was 249.7 °C and was decreased to 229.84 °C when mixed evenly with Kollidon(®) MAE 100P. However, the melting point was not detected at a ratio of 1:4 since it fully dissolved or dispersed in the polymer. Glass transition temperature (T g ) of different compositions exhibited strong interaction of polymer and drug. The result was supported by spectra evidence that Kollidon(®) MAE 100P forms hydrogen bonds with dutasteride presenting strong physical interaction with the primary amine group of dutasteride. This study supports a convenient method that together with microscopic observation can perform polymer selection and characterize solid dispersions.

Preparation of microcapsules with the evaluation of physicochemical properties and molecular interaction.

The objective of this study was to prepare and characterize dutasteride (a hydrophobic model drug) microcapsules using ethyl cellulose as a capsule shell polymer with different drug/polymer ratios of 1:1, 1:3, and 1:5. The microcapsules were prepared by a solvent evaporation method and the prepared microcapsules were evaluated for percent yield, percent drug content, encapsulation efficiency, particle size distribution, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), and in vitro drug release studies. SEM revealed the spherical shape of all prepared microcapsules. The particle size of the microcapsules was about 95-119 µm with good yield and encapsulation efficiency. PXRD showed different X-ray patterns compared to the drug itself suggesting possibility of crystalline form change during the process. Moreover, it confirmed that ethyl cellulose was changed to amorphous state. The physical property changes may affect the overall quality and drug release behavior. In the FT-IR studies, hydrogen bonding was observed between the drug and polymer at the molecular level. DSC data provided consistent results with the FT-IR and PXRD analyses. Drug release profiles showed the overall sustained release of drug and anomalous diffusion mechanism based on the Korsmeyer-Peppas equation. Understanding the physicochemical properties of a drug and polymer including molecular interactions may facilitate formulation of microcapsules with acceptable properties and drug release behaviors.

Synthesis and characterization of 3ß-substituted amides of 17a-aza-D-homo- 4-androsten-17-one as potent 5α-reductase inhibitors and antimicrobial agents.

We herein report the synthesis of 3ß-substituted amides of 17a-aza-D-homo-4-androsten-17-one (11a-11r) from commercially available Diosgenin as the starting material. The structures of newly synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR and mass spectrometry. All the synthesized analogues were tested for their 5α- reductase inhibitory and antimicrobial activity, some of them exhibit moderate to potent activity comparable to the reference drugs. Among the synthesized derivatives the analogue (11r) 3ß-(indonlylbutanamido)-17a-aza-D-homo-4- androsten-17-one was found to be active against both 5α-reductase enzyme and microbial strains, whereas the analogue (11i) 3ß-(3,4-dimethoxy-benzamido)-17a-aza-D-homo-4-androsten-17-one was found to be the least active. The detailed 5α-reductase inhibitors and antimicrobial activities of the synthesized compounds were reported.

Influence of hydrophilic additives on the supersaturation and bioavailability of dutasteride-loaded hydroxypropyl-ß-cyclodextrin nanostructures.

The objectives of this study were to develop a novel solid dutasteride formulation with improved physicochemical properties and oral bioavailability, and to examine the correlation between its in vitro dissolution and in vivo pharmacokinetic parameters. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) nanostructures with or without hydrophilic additives were manufactured using the supercritical antisolvent process. The dutasteride-loaded HP-ß-CD nanoparticles formed aggregates with a mean particle size of less than 160 nm and a specific surface area greater than 100 m(2)/g. Increases in the supersaturation and dissolution rate for dutasteride were dependent on the type of additive; increases in maximum solubility and extended supersaturation were observed in dutasteride-loaded HP-ß-CD nanostructures with hydroxypropylmethyl cellulose, whereas the dissolution rate was the highest for nanostructures containing d-α-tocopheryl polyethylene glycol 1000 succinate. In rats, the oral bioavailability of dutasteride increased with the supersaturation induced by the HP-ß-CD nanostructures. In addition, compared with the in vitro drug release rate, the in vivo pharmacokinetic parameters were more closely correlated with in vitro parameters related to supersaturation (solubility). Further, the bioavailability of the dutasteride-loaded HP-ß-CD nanostructures with hydroxypropylmethyl cellulose was similar to that of the commercially available soft gelatin capsule (Avodart®). In conclusion, preparation of dutasteride-loaded HP-ß-CD nanostructures using the supercritical antisolvent process affords a viable alternative solid dosage form for dutasteride.

Reliable and sensitive determination of dutasteride in human plasma by liquid chromatography-tandem mass spectrometry.

An accurate and precise method was developed and validated using LC-MS/MS to quantify dutasteride in human plasma. The analyte and dutasteride-13C6 as internal standard (IS) were extracted from 300 µL plasma volume using methyl tert-butyl ether-n-hexane (80:20, v/v). Chromatographic analysis was performed on a Gemini C18 (150 × 4.6 mm, 5 µm) column using acetonitrile-5 mm ammonium formate, pH adjusted to 4.0 with formic acid (85:15, v/v) as the mobile phase. Tandem mass spectrometry in positive ionization mode was used to quantify dutasteride by multiple reaction monitoring. The entire data processing was done using Watson LIMS(TM) software, which provided excellent data integrity and high throughput with improved operational efficiency. The calibration curve was linear in the range of 0.1-25 ng/mL, with intra-and inter-batch values for accuracy and precision (coefficient of variation) ranging from 95.8 to 104.0 and from 0.7 to 5.3%, respectively. The mean overall recovery across quality controls was ≥95% for the analyte and IS, while the interference of matrix expressed as IS-normalized matrix factors ranged from 1.01 to 1.02. The method was successfully applied to support a bioequivalence study of 0.5 mg dutasteride capsules in 24 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 103 incurred samples.