Steroid and Triterpenoid Compounds with Antiparasitic Properties.

Parasitic diseases affect millions of people and animals, predominantly in the tropics, including visitors to tropical countries and other areas. Efficient and low-cost treatments for infections caused by various parasites are not yet available. Antiparasitic drugs have some drawbacks, such as toxicity and the development of resistance by parasites. This has motivated many researchers to focus on the discovery of safe, effective and affordable antiparasitic drugs, both among drugs already available for other diseases and new compounds synthesized or isolated from natural sources. Furthermore, steroid and triterpenoid compounds attract the attention of pharmacologists, chemists and biochemists owing to their broad application in the treatment of various diseases. Isolation of steroid and triterpenoid compounds from natural sources with antiparasitic efficacy is an attractive choice for scientists. On the other hand, these compounds can be transformed into more potent forms by modifying the basic skeleton. This review presents a collection of isolated and synthesized steroid and triterpenoid compounds from 2018 to 2021 that have been reported to be effective against certain parasitic protozoa and helminths. A total of 258 compounds have been identified with antimalarial, antitrypanosomal, antileishmanial, anti-Toxoplasma, and/or anthelmintic activity. The described investigations of antiparasitic compounds may be helpful for further drug development.

Chemometrics of anisotropic lipophilicity of anticancer androstane derivatives determined by reversed-phase ultra high performance liquid chromatography with polar aprotic and protic modifiers.

The research of the use of steroid compounds in the treatment of various types of cancer has a history of more than 50 years. Numerous steroid derivatives have expressed significant anticancer activity, however the thorough analysis of their physicochemical and toxicological properties is required prior to their clinical application. The present study is focused on the characterization of physicochemical properties of a series of previously synthesized new androstane derivatives (16E-hydroxyimino derivatives, D-homo lactones and D-seco dinitriles) with anticancer activity applying reversed-phase ultra high performance liquid chromatography (RP-UHPLC) system under isocratic conditions and chemometric tools, including in silico determination of their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The chromatographic analysis was carried out applying two two-component and one ternary mobile phases with aprotic (acetonitrile) and protic (water and methanol) solvents. The conducted quantitative structure-retention relationship modeling resulted in two univariate and nine multivariate linear mathematical models that successfully correlate the retention parameters (logk) with descriptors of molecular polarity/lipophilicity (tPSA, REF, Average LogP, ClogP, ALOGP, LogS-SILICOS-IT, AClogS), descriptors that mainly depend on intermolecular interactions (BP, CP, CT, DE, HL) and ADMET descriptors (SWISSLogKpSP, GPCR, HIA, EI). The quality of the models was proved by the internal and external validation, while the robustness of the models was confirmed by Y-randomization test. Considering the established meaningful relationships between physicochemical/ADMET properties and retention parameters, the determined logk parameters of the analyzed series of steroid derivatives can be considered to be a biopharmaceutical property from the perspective of lipophilicity.

Unraveling the anti-virulence potential and antifungal efficacy of 5-aminotetrazoles using the zebrafish model of disseminated candidiasis.

Candida albicans remains the main causal agent of candidiasis, the most common fungal infection with disturbingly high mortality rates worldwide. The limited diversity and efficacy of clinical antifungal drugs, exacerbated by emerging drug resistance, have resulted in the failure of current antifungal therapies. This imposes an urgent demand for the development of innovative strategies for effective eradication of candidal infections. While the existing clinical drugs display fungicidal or fungistatic activity, the strategy specifically targeting C. albicans filamentation, as the most important virulence trait, represents an attractive approach for overcoming the drawbacks related to clinical antifungals. The results acquired in this study revealed the significant potential of 5-aminotetrazoles as a new class of effective and safe anti-virulence agents. Moreover, these novel agents were active when applied both alone and in combination with clinically approved polyenes. Complete prevention of C. albicans morphogenetic yeast-to-hyphae transition was achieved at doses as low as 1.3 µM under conditions mimicking various filamentation-responsive stimuli in the human body, while no cardio- or hepatotoxicity was observed at doses as high as 200 µM. The treatment of C. albicans-infected zebrafish embryos with nystatin alone had low efficacy, while the combination of nystatin and selected 5-aminotetrazoles prevented fungal filamentation, successfully eliminating the infection and rescuing the infected embryos from lethal disseminated candidiasis. In addition, the most potent anti-virulence 5-aminotetrazole prevented C. albicans in developing the resistance to nystatin when applied in combination, keeping the fungus sensitive to the antifungal drug.

Heterocyclic androstane and estrane d-ring modified steroids: Microwave-assisted synthesis, steroid-converting enzyme inhibition, apoptosis induction, and effects on genes encoding estrogen inactivating enzymes.

d-ring-fused and d-homo lactone compounds in estratriene and androstane series were synthesized using microwave-assisted reaction conditions. Microwave-irradiated synthesis methods were convenient and effective, and provided high yields with short reaction times. Their inhibition of C17,20-lyase and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities were studied in in vitro enzyme assays. d-ring-fused triazolyl estrone analog 24 showed potent inhibition of NADH-complexed 17ß-HSD1, with a binding affinity similar to that of the substrate estrone; its inhibition against NADPH-complexed 17ß-HSD1 was markedly weaker. Compound 24 also significantly and selectively reduced proliferation of cancer cell lines of gynecological origin. This estrane triazole changed the cell cycle and induced apoptosis of HeLa, SiHa, and MDA-MB-231 cancer cells, measured by both increased subG1 fraction of cells and activation of caspase-independent signaling pathways. A third mode of anti-estrogenic action of 24 saw increased mRNA expression of the SULT1E1 gene in HeLa cells; in contrast, its 3-benzyloxy analog 23 increased mRNA expression of the HSD17B2 gene, thus showing pronounced pro-drug anti-estrogenic activity. Estradiol-derived d-ring triazole compound 24 thus acts at the enzyme, gene expression and cellular levels to decrease the production of active estrogen hormones, demonstrating its pharmacological potential.

Controlling Pd-Catalyzed N-Arylation and Dimroth Rearrangement in the Synthesis of N,1-Diaryl-1H-tetrazol-5-amines.

The Pd-catalyzed N-arylation method for the synthesis of eighteen N,1-diaryl-1H-tetrazol-5-amine derivatives is reported. By running the reactions at 35 °C, compounds were isolated as single isomers since the undesired Dimroth rearrangement was completely suppressed. Furthermore, the Dimroth rearrangement of N,1-diaryl-1H-tetrazol-5-amines was rationalized by conducting comprehensive experiments and NMR analysis as well as density functional theory (DFT) calculations of thermodynamic stability of the compounds. It was established that the Dimroth rearrangement is thermodynamically controlled, and the equilibrium of the reaction is determined by the stability of the corresponding isomers. The mechanism was investigated by additional DFT calculations, and the opening of the tetrazole ring was shown to be the rate-determining step. By maneuvering Pd-catalyzed N-arylation and the subsequent Dimroth rearrangement, two more N,1-diaryl-1H-tetrazol-5-amine derivatives were acquired, which otherwise cannot be synthesized by employing the C-N cross-coupling reaction.

Heat-induced longevity in budding yeast requires respiratory metabolism and glutathione recycling.

Heat-induced hormesis is a well-known conserved phenomenon in aging, traditionally attributed to the benefits conferred by increased amounts of heat shock (HS) proteins. Here we find that the key event for the HS-induced lifespan extension in budding yeast is the switch from glycolysis to respiratory metabolism. The resulting increase in reactive oxygen species activates the antioxidant response, supported by the redirection of glucose from glycolysis to the pentose phosphate pathway, increasing the production of NADPH. This sequence of events culminates in replicative lifespan (RLS) extension, implying decreased mortality per generation that persists even after the HS has finished. We found that switching to respiratory metabolism, and particularly the consequent increase in glutathione levels, were essential for the observed RLS extension. These results draw the focus away solely from the HS response and demonstrate that the antioxidant response has a key role in heat-induced hormesis. Our findings underscore the importance of the changes in cellular metabolic activity for heat-induced longevity in budding yeast.

Anticancer activity of novel steroidal 6-substituted 4-en-3-one D-seco dinitriles.

Steroidal 16,17-seco-16,17a-dinitriles possessing 4-ene-3,6-dione (3), 6-methylene-4-en-3-one (5), (6E)-hydroxyimino-4-en-3ß-ol (9) or (6E)-hydroxyimino-4-en-3-one (10) moiety were synthesized starting from 3ß-acetoxy-16,17-secoandrost-4-ene-16,17a-dinitrile (1). Antiproliferative activity of the newly synthesized compounds, as well as previously synthesized 3-oxo-16,17-secoandrosta-1,4-diene-16,17a-dinitrile (VII), was tested in vitro. Compound 9 displayed submicromolar antiproliferative activity against human cervical carcinoma (HeLa) cells (IC50 0.48 µM), and compounds 3 and 10 expressed strong inhibitory potential against HeLa cells (IC50 4.31 µM and 2.64 µM, respectively). Also, compound 10 was effective in inhibiting estrogen hormone-independent (MDA-MB-231) cells (IC50 2.78 µM). All tested compounds had no influence on the proliferation of healthy cells (MRC-5). Since MDA-MB-231 breast cancer cells and HeLa cervical cancer cells were most sensitive to treatment by 16,17-seco-16,17a-dinitriles, apoptosis induction after treatment by compounds 3, VII, 9 and 10 was studied in these cells, to reveal the mechanism underlying cell growth inhibition. All tested compounds significantly induced apoptosis in both treated cell lines, which was evident from results obtained by a double AO-EB staining test and quantified by counting cells with apoptotic morphology after staining with Giemsa dye. Among all tested substances, (6E)-hydroxyimino-4-en-3-one derivative 10 expressed the most proapoptotic activity.

Identification of d-seco modified steroid derivatives with affinity for estrogen receptor α and ß isoforms using a non-transcriptional fluorescent cell assay in yeast.

Synthesis and biological evaluation of steroidal derivatives with anticancer properties is an active area of drug discovery. Here we measured the relative affinities of d-seco modified steroidal derivatives for estrogen receptor α, estrogen receptor ß or androgen receptor ligand binding domains using an optimized non-transcriptional fluorescent cell assay in yeast. Ligand binding domains of steroid receptors were expressed in-frame with yellow fluorescent protein in the yeast Saccharomyces cerevisiae. Addition of known steroid ligands to yeast expressing the appropriate cognate receptor results in increased fluorescence intensity, enabling estimation of receptor binding affinities in a dose-response and time-dependent manner. Relative binding affinities of d-seco modified steroidal derivatives 1-4 were then evaluated using this yeast system by live cell fluorimetry and fluorescence microscopy, coupled with in vitro cytotoxicity and in silico molecular docking studies. d-Seco estratriene derivative 2displayed strong affinity for both estrogen receptor α and ß ligand binding domains and negligible affinity for the androgen receptor ligand binding domain. Compound 2 also showed moderate cytotoxicity against estrogen receptor positive MCF-7 breast adenocarcinoma cells. In addition to identification of new ligands for steroid receptors, this assay could also be used to filter out compounds with potential for off-target interactions with steroid receptors during the early stages of compound screening.

TORC1-mediated sensing of chaperone activity alters glucose metabolism and extends lifespan.

Protein quality control mechanisms, required for normal cellular functioning, encompass multiple functions related to protein production and maintenance. However, the existence of communication between proteostasis and metabolic networks and its underlying mechanisms remain elusive. Here, we report that enhanced chaperone activity and consequent improved proteostasis are sensed by TORC1 via the activity of Hsp82. Chaperone enrichment decreases the level of Hsp82, which deactivates TORC1 and leads to activation of Snf1/AMPK, regardless of glucose availability. This mechanism culminates in the extension of yeast replicative lifespan (RLS) that is fully reliant on both TORC1 deactivation and Snf1/AMPK activation. Specifically, we identify oxygen consumption increase as the downstream effect of Snf1 activation responsible for the entire RLS extension. Our results set a novel paradigm for the role of proteostasis in aging: modulation of the misfolded protein level can affect cellular metabolic features as well as mitochondrial activity and consequently modify lifespan. The described mechanism is expected to open new avenues for research of aging and age-related diseases.

Chemometrics approach based on chromatographic behavior, in silico characterization and molecular docking study of steroid analogs with biomedical importance.

Physicochemical characterization of steroid analogs (triazole, tetrazole, toluenesulfonylhydrazide, nitrile, dinitrile and dione) is considered to be a very important step in further drug selection. This study applies to the determination of lipophilicity of previously synthesized steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC). Chemometric aspect of chromatographic lipophilicity is given throughout multiple linear regression (MLR) quantitative structure-retention relationships (QSRR) approach. Minimal inhibitory concentration (MIC) is determined for two steroid derivatives possessing antimicrobial activity against Staphylococcus aureus. Molecular docking study was performed in order to identify the compound with the most promising potential as human cytochrome P450 CYP17A1inhibitor. Identified 3ß-hydroxyandrost-5-eno[16,17-d]-1,2,3-triazole (I.2.) could be recommended for further trials for anticancer drugs and subjected to the absorption, distribution, metabolism, excretion and toxicity (ADMET) evaluation.

Lipophilicity estimation and characterization of selected steroid derivatives of biomedical importance applying RP HPLC.

The present paper deals with chromatographic lipophilicity determination of twenty-nine selected steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC) combined with two mobile phase, acetonitrile-water and methanol-water. Chromatographic behavior of four groups (triazole and tetrazole, toluenesulfonylhydrazide, nitrile and dinitrile and dione) of selected steroid derivatives was studied. Investigated compounds were grouped using principal component analysis (PCA) according to their logk values for both mobile phases. Grouping was in the very good accordance with the polarity and lipophilicity of the investigated compounds. QSRR (quantitative structure-retention relationship) approach was used to model chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods. The most influential molecular descriptors were lipophilicity descriptors that are important for molecules ability to pass through biological membranes and geometrical descriptors. All established LR-QSRR and MLR-QSRR models were statistically validated by standards, cross- and external validation parameters as well as with two graphical methods. According to all these assessments, MLR models were better for chromatographic lipophilicity prediction. It was shown that chromatographic systems with methanol-water were better for modeling of logk than systems with acetonitrile-water, as well as the systems that contained lower volume fractions of organic component in mobile phase. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates of biomedical importance.

Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan.

In cells living under optimal conditions, protein folding defects are usually prevented by the action of chaperones. Here, we investigate the cell-wide consequences of loss of chaperone function in cytosol, mitochondria or the endoplasmic reticulum (ER) in budding yeast. We find that the decline in chaperone activity in each compartment results in loss of respiration, demonstrating the dependence of mitochondrial activity on cell-wide proteostasis. Furthermore, each chaperone deficiency triggers a response, presumably via the communication among the folding environments of distinct cellular compartments, termed here the cross-organelle stress response (CORE). The proposed CORE pathway encompasses activation of protein conformational maintenance machineries, antioxidant enzymes, and metabolic changes simultaneously in the cytosol, mitochondria, and the ER. CORE induction extends replicative and chronological lifespan in budding yeast, highlighting its protective role against moderate proteotoxicity and its consequences such as the decline in respiration. Our findings accentuate that organelles do not function in isolation, but are integrated in a functional crosstalk, while also highlighting the importance of organelle communication in aging and age-related diseases.

Synthesis and anticancer cell potential of steroidal 16,17-seco-16,17a-dinitriles: identification of a selective inhibitor of hormone-independent breast cancer cells.

We report the synthesis of steroidal 16,17-seco-16,17a-dinitriles and investigate their antitumor cell properties. Compounds were evaluated for anticancer potential by in vitro antiproliferation studies, molecular docking and virtual screening. Several compounds inhibit the growth of breast and prostate cancer cell lines (MCF-7, MDA-MB-231 and PC3), and/or cervical cancer cells (HeLa). Supporting this, molecular docking predicts that steroidal 16,17-seco-16,17a-dinitriles could bind with high affinity to multiple molecular targets of breast and prostate cancer treatment (aromatase, estrogen receptor α, androgen receptor and 17α-hydroxylase) facilitated by D-seco flexibility and nitrile-mediated contacts. Thus, 16,17-seco-16,17a-dinitriles may be useful for the design of inhibitors of multiple steroidogenesis pathways. Strikingly, 10, a 1,4-dien-3-on derivative, displayed selective submicromolar antiproliferative activity against hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells (IC50 0.52, 0.11µM, respectively). Ligand-based 3D similarity searches suggest AKR1C, 17ß-HSD and/or 3ß-HSD subfamilies as responsible for this antiproliferative activity, while fast molecular docking identified AKR1C and ERß as potential binders-both targets in the treatment of hormone-independent breast cancers.

Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives.

A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ(5)-3ßHSD, 17ßHSD2 and 17ßHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17ßHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant.

Signatures of conformational stability and oxidation resistance in proteomes of pathogenic bacteria.

Protein oxidation is known to compromise vital cellular functions. Therefore, invading pathogenic bacteria must resist damage inflicted by host defenses via reactive oxygen species. Using comparative genomics and experimental approaches, we provide multiple lines of evidence that proteins from pathogenic bacteria have acquired resistance to oxidative stress by an increased conformational stability. Representative pathogens exhibited higher survival upon HSP90 inhibition and a less-oxidation-prone proteome. A proteome signature of the 46 pathogenic bacteria encompasses 14 physicochemical features related to increasing protein conformational stability. By purifying ten representative proteins, we demonstrate in vitro that proteins with a pathogen-like signature are more resistant to oxidative stress as a consequence of their increased conformational stability. A compositional signature of the pathogens' proteomes allowed the design of protein fragments more resilient to both unfolding and carbonylation, validating the relationship between conformational stability and oxidability with implications for synthetic biology and antimicrobial strategies.