Transcriptional Inhibition of MicroRNA miR-122 by Small Molecules Reduces Hepatitis C Virus Replication in Liver Cells.

MicroRNAs (miRNAs) are noncoding RNA molecules of 22-24 nucleotides that are estimated to regulate thousands of genes in humans, and their dysregulation has been implicated in many diseases. MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and has been linked to the development of hepatocellular carcinoma and hepatitis C virus (HCV) infection. Its role in these diseases renders miR-122 a potential target for small-molecule therapeutics. Here, we report the discovery of a new sulfonamide class of small-molecule miR-122 inhibitors from a high-throughput screen using a luciferase-based reporter assay. Structure-activity relationship (SAR) studies and secondary assays led to the development of potent and selective miR-122 inhibitors. Preliminary mechanism-of-action studies suggest a role in the promoter-specific transcriptional inhibition of miR-122 expression through direct binding to the liver-enriched transcription factor hepatocyte nuclear factor 4α. Importantly, the developed inhibitors significantly reduce HCV replication in human liver cells.

Motivations, dating app relationships, unintended consequences and change in sexual behaviour in dating app users at an Australian music festival.

BACKGROUND: Despite the popularity of dating apps, there remain scarce data on the motivations, consequences and their influence on sexual behaviour change in the Australian population. OBJECTIVE: To explore motivations, dating app relationships, unintended consequences and change in sexual behaviour in dating app users at an Australian music festival. METHODS: A cross-sectional study design was used. Festival patrons aged 18-30 at a major Australian music festival completed a paper-based survey. Logistic regression was used to identify which factors were associated with an increase in sexual partners since using dating apps. RESULTS: The primary reasons for dating app use (N = 437) were boredom (59.7%), casual sex (45.1%) and casual dating (42.8%). A third of users used them at music festivals (33.8%, n = 432). A third of participants had used dating apps for more than 2 years (33.3%) and a third (33.0%) of users claimed to have changed their sexual behaviour after app use, including increased frequency of sexual activity (70.0%), number of sexual partners (57.1%) and sexual experimentation (42.1%). Dating app users tended not to discuss sexually transmitted infections (STI) status with a sexual partner regardless of whether they had met them on an app or not: 38.5% would 'never' and 36.9% would 'sometimes' have safe sex discussions with partners met via apps. Condoms were 'always' used for 36.9% of dating app users when meeting partners via dating apps, compared to 29.9% met by other means. 8.6% of dating app users reported having contracted STIs, and 2.8% had unwanted pregnancies with those met on dating apps. After adjusting for socio-demographics, those who had an STI after engaging in sexual activity with a person met via a dating app had 2.4 times the odds of reporting an increase in sexual partners, and those who had used a dating app for over 2 years had twice the odds of reporting an increase in sexual partners. When condom use was entered into the model, those that 'often' or 'sometimes' used a condom with a new dating app partner were twice as likely to report an increase in sexual partners since using dating apps, compared to those who 'always' used a condom with a new dating app partner. Sexual orientation and STI discussions with a new sexual dating app partner were not associated with an increase in dating app partners. CONCLUSION: Dating app usage is common and users report increased sexual activity, sexual partners and experimentation. STI discussions with potential partners and condom use remained low regardless of how partners were met and despite an increase in sexual partners since using dating apps. Given the high-risk nature of individuals that utilise dating apps, safe sex discussion, including STIs, pregnancies and condom use should be promoted to improve sexual health outcomes.

Geosocial Networking Dating App Usage and Risky Sexual Behavior in Young Adults Attending a Music Festival: Cross-sectional Questionnaire Study.

BACKGROUND: Despite the prevalent use of geosocial networking dating apps (GNDAs), there is limited research on their impact on sexual health outcomes among young music festivals attendees. OBJECTIVE: This study aims to explore the use of GNDAs and risky sexual behaviors of young adults attending a music festival. METHODS: The music festival attendees (N=862) completed a cross-sectional questionnaire study encompassing demographics, dating app use, and risky sexual behaviors in the past year. Associations between these variables were estimated using bivariate and multivariate logistic regression analyses. RESULTS: Of the respondents, 51.9% (448/862) had used GNDAs in the previous year. Compared with people who had 1 partner, people who had 2-5 sexual partners in the previous year had almost 7 times the odds of using dating apps (odds ratio [OR] 6.581, 95% CI 4.643-9.328) and those who had more than 5 partners had 14 times the odds of using dating apps (OR 14.294, 95% CI 8.92-22.906). Condom users were more likely to be app users (P<.001), as were those who relied on emergency Plan B (P=.002), but people using hormonal contraception were less likely to use dating apps (P=.004). After adjusting for sexual orientation and relationship status, those having casual sex had 3.096 (95% CI 2.225-4.307; P<.001) times the odds of using dating apps and those having multiple sexual partners had 3.943 (95% CI 2.782-5.588; P<.001) times the odds of using dating apps. Similarly, after adjusting for sexual orientation, relationship status, and number of sexual partners, people who had no discussions before having sex about sexually transmitted infections (STIs) or boundaries were more likely to use dating apps (OR 1.755, 95% CI 1.232-2.500; P=.002). Those who perceived the risk of having sex without contraception to be very high had 2.486 (95% CI 2.213-5.096; P=.01) times the odds of using dating apps than those who perceived no risk. Compared with those who perceived no risk, people who thought that the risk of having multiple sexual partners was low to high had 1.871 (95% CI 1.024-3.418; P=.04) times the odds of using dating apps. A significant number of app users (389/440, 88.4%) indicated that GNDAs should promote safe sex. CONCLUSIONS: This study identified that festival goers engaging in certain high-risk sexual behaviors, including casual sex, having multiple sexual partners, and having sex without discussion about STI status and boundaries, are more likely to use dating apps. Festival goers who perceived sex without any form of contraception, having sex while drunk, and having multiple sexual partners as risky were more likely to be app users. Policy makers and GNDA developers should acknowledge the vulnerability of their users to adverse sexual health outcomes and use GNDAs as a platform to promote risk-reduction practices.

Small molecule inhibition of microRNA-21 expression reduces cell viability and microtumor formation.

MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of a large number of protein-coding genes and are implicated in a variety of biological processes such as development, differentiation, proliferation, and cell survival. Dysregulation of miRNAs has been attributed to the onset and progression of various human diseases, including cancer. MicroRNA-21 (miR-21), one of the most established oncogenic miRNAs, is found to be upregulated in a wide range of cancers making it an attractive therapeutic target. Employment of a luciferase-based live-cell reporter assay in a high-throughput screen of >300,000 small molecules led to the discovery of a new class of ether-amide miR-21 inhibitors. Following a structure-activity relationship study, an optimized lead molecule was found to inhibit miR-21 transcription. Furthermore, the inhibitor demonstrated cytotoxicity in a cervical cancer cell line via induction of apoptosis and was capable of reducing microtumor formation in a long-term clonogenic assay. Altogether, this work reports the discovery of a new small molecule inhibitor of miR-21 and demonstrates its potential as an alternative approach in cancer therapy.

Small Molecule Inhibition of MicroRNA miR-21 Rescues Chemosensitivity of Renal-Cell Carcinoma to Topotecan.

Chemical probes of microRNA (miRNA) function are potential tools for understanding miRNA biology that also provide new approaches for discovering therapeutics for miRNA-associated diseases. MicroRNA-21 (miR-21) is an oncogenic miRNA that is overexpressed in most cancers and has been strongly associated with driving chemoresistance in cancers such as renal cell carcinoma (RCC). Using a cell-based luciferase reporter assay to screen small molecules, we identified a novel inhibitor of miR-21 function. Following structure-activity relationship studies, an optimized lead compound demonstrated cytotoxicity in several cancer cell lines. In a chemoresistant-RCC cell line, inhibition of miR-21 via small molecule treatment rescued the expression of tumor-suppressor proteins and sensitized cells to topotecan-induced apoptosis. This resulted in a >10-fold improvement in topotecan activity in cell viability and clonogenic assays. Overall, this work reports a novel small molecule inhibitor for perturbing miR-21 function and demonstrates an approach to enhancing the potency of chemotherapeutics specifically for cancers derived from oncomir addiction.

Activity of caffeic acid phenethyl ester in Caenorhabditis elegans.

AIM: Caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE), the major constituent of propolis, is able to increase the survival of the nematode Caenorhabditis elegans after infection with the fungal pathogen Candida albicans. RESULTS: CAPE increases the expression of several antimicrobial proteins involved in the immune response to C. albicans. Structural derivatives of CAPE were synthesized to identify structure-activity relationships and decrease metabolic liability, ultimately leading to a compound that has similar efficacy, but increased in vivo stability. The CED-10(Rac-1)/PAK1 pathway was essential for immunomodulation by CAPE and was a critical component involved in the immune response to fungal pathogens. CONCLUSION: Caenorhabditis elegans is an efficient heterologous host to evaluate immunomodulatory compounds and identify components of the pathway(s) involved in the mode of action of compounds.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

Aryl amide small-molecule inhibitors of microRNA miR-21 function.

MicroRNAs (miRNAs) are single stranded RNA molecules of ∼22 nucleotides that negatively regulate gene expression. MiRNAs are involved in fundamental cellular processes, such as development, differentiation, proliferation, and survival. MiRNA misregulation has been linked to various human diseases, most notably cancer. MicroRNA-21 (miR-21), a well-established oncomiR, is significantly overexpressed in many types of human cancers, thus rendering miR-21 a potential therapeutic target. Using a luciferase-based reporter assay under the control of miR-21 expression, a high-throughput screen of >300,000 compounds led to the discovery of a new aryl amide class of small-molecule miR-21 inhibitors. Structure-activity relationship (SAR) studies resulted in the development of four aryl amide derivatives as potent and selective miR-21 inhibitors. The intracellular levels of various miRNAs in HeLa cells were analyzed by qRT-PCR revealing specificity for miR-21 inhibition over other miRNAs. Additionally, preliminary mechanism of action studies propose a different mode of action compared to previously reported miR-21 inhibitors, thus affording a new chemical probe for future studies.

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides.

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.

Cellular delivery and photochemical activation of antisense agents through a nucleobase caging strategy.

Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is typically needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent, and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity.

Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif.

Hydroxamic acids were designed, synthesized, and evaluated for their ability to selectively inhibit human histone deacetylase 6 (HDAC6). Several inhibitors, including compound 14 (BRD9757), exhibited excellent potency and selectivity despite the absence of a surface-binding motif. The binding of these highly efficient ligands for HDAC6 is rationalized via structure-activity relationships. These results demonstrate that high selectivity and potent inhibition of HDAC6 can be achieved through careful choice of linker element only.

Synthesis and profiling of a diverse collection of azetidine-based scaffolds for the development of CNS-focused lead-like libraries.

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function.

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3' untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.