Development of VPC-70619, a Small-Molecule N-Myc Inhibitor as a Potential Therapy for Neuroendocrine Prostate Cancer.

The Myc family of transcription factors are involved in the development and progression of numerous cancers, including prostate cancer (PCa). Under the pressure of androgen receptor (AR)-directed therapies resistance can occur, leading to the lethal form of PCa known as neuroendocrine prostate cancer (NEPC), characterized among other features by N-Myc overexpression. There are no clinically approved treatments for NEPC, translating into poor patient prognosis and survival. Therefore, there is a pressing need to develop novel therapeutic avenues to treat NEPC patients. In this study, we investigate the N-Myc-Max DNA binding domain (DBD) as a potential target for small molecule inhibitors and utilize computer-aided drug design (CADD) approaches to discover prospective hits. Through further exploration and optimization, a compound, VPC-70619, was identified with notable anti-N-Myc potency and strong antiproliferative activity against numerous N-Myc expressing cell lines, including those representing NEPC.

Computer-Aided Ligand Discovery for Estrogen Receptor Alpha.

Breast cancer (BCa) is one of the most predominantly diagnosed cancers in women. Notably, 70% of BCa diagnoses are Estrogen Receptor α positive (ERα+) making it a critical therapeutic target. With that, the two subtypes of ER, ERα and ERß, have contrasting effects on BCa cells. While ERα promotes cancerous activities, ERß isoform exhibits inhibitory effects on the same. ER-directed small molecule drug discovery for BCa has provided the FDA approved drugs tamoxifen, toremifene, raloxifene and fulvestrant that all bind to the estrogen binding site of the receptor. These ER-directed inhibitors are non-selective in nature and may eventually induce resistance in BCa cells as well as increase the risk of endometrial cancer development. Thus, there is an urgent need to develop novel drugs with alternative ERα targeting mechanisms that can overcome the limitations of conventional anti-ERα therapies. Several functional sites on ERα, such as Activation Function-2 (AF2), DNA binding domain (DBD), and F-domain, have been recently considered as potential targets in the context of drug research and discovery. In this review, we summarize methods of computer-aided drug design (CADD) that have been employed to analyze and explore potential targetable sites on ERα, discuss recent advancement of ERα inhibitor development, and highlight the potential opportunities and challenges of future ERα-directed drug discovery.

Dual-Inhibitors of N-Myc and AURKA as Potential Therapy for Neuroendocrine Prostate Cancer.

Resistance to androgen-receptor (AR) directed therapies is, among other factors, associated with Myc transcription factors that are involved in development and progression of many cancers. Overexpression of N-Myc protein in prostate cancer (PCa) leads to its transformation to advanced neuroendocrine prostate cancer (NEPC) that currently has no approved treatments. N-Myc has a short half-life but acts as an NEPC stimulator when it is stabilized by forming a protective complex with Aurora A kinase (AURKA). Therefore, dual-inhibition of N-Myc and AURKA would be an attractive therapeutic avenue for NEPC. Following our computer-aided drug discovery approach, compounds exhibiting potent N-Myc specific inhibition and strong anti-proliferative activity against several N-Myc driven cell lines, were identified. Thereafter, we have developed dual inhibitors of N-Myc and AURKA through structure-based drug design approach by merging our novel N-Myc specific chemical scaffolds with fragments of known AURKA inhibitors. Favorable binding modes of the designed compounds to both N-Myc and AURKA target sites have been predicted by docking. A promising lead compound, 70812, demonstrated low-micromolar potency against both N-Myc and AURKA in vitro assays and effectively suppressed NEPC cell growth.

Benzothiophenone Derivatives Targeting Mutant Forms of Estrogen Receptor-α in Hormone-Resistant Breast Cancers.

Estrogen receptor-α positive (ERα⁺) breast cancers represent 75% of all invasive breast cancer cases, while de novo or acquired resistance to ER-directed therapy is also on the rise. Numerous factors contribute to this phenomenon including the recently-reported ESR1 gene mutations such as Y537S, which amplifies co-activator interactions with ERα and promotes constitutive activation of ERα function. Herein, we propose that direct targeting of the activation function-2 (AF2) site on ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. A systematic computer-guided drug discovery approach was employed to develop a potent ERα inhibitor that was extensively evaluated by a series of experiments to confirm its AF2-specific activity. We demonstrate that the developed small-molecule inhibitor effectively prevents ERα-coactivator interactions and exhibits a strong anti-proliferative effect against tamoxifen-resistant cells, as well as downregulates ERα-dependent genes and effectively diminishes the receptor binding to chromatin. Notably, the identified lead compound successfully inhibits known constitutively-active, resistance-associated mutant forms of ERα observed in clinical settings. Overall, this study reports the development of a novel class of ERα AF2 inhibitors, which have the potential to effectively inhibit ERα activity by a unique mechanism and to circumvent the issue of mutation-driven resistance in breast cancer.

Computer-Aided Discovery of Small Molecule Inhibitors of Transcriptional Activity of TLX (NR2E1) Nuclear Receptor.

Orphan nuclear receptor TLX (NR2E1) plays a critical role in the regulation of neural stem cells (NSC) as well as in the development of NSC-derived brain tumors. In the last years, new data have emerged implicating TLX in prostate and breast cancer. Therefore, inhibitors of TLX transcriptional activity may have a significant impact on the treatment of several critical malignancies. However, the TLX protein possesses a non-canonical ligand-binding domain (LBD), which lacks a ligand-binding pocket (conventionally targeted in case of nuclear receptors) that complicates the development of small molecule inhibitors of TLX. Herein, we utilized a rational structure-based design approach to identify small molecules targeting the Atro-box binding site of human TLX LBD. As a result of virtual screening of ~7 million molecular structures, 97 compounds were identified and evaluated in the TLX-responsive luciferase reporter assay. Among those, three chemicals demonstrated 40⁻50% inhibition of luciferase-detected transcriptional activity of the TLX orphan nuclear receptor at a dose of 35 µM. The identified compounds represent the first class of small molecule inhibitors of TLX transcriptional activity identified via methods of computer-aided drug discovery.

Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer.

The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

An enzyme captured in two conformational states: crystal structure of S-adenosyl-L-homocysteine hydrolase from Bradyrhizobium elkanii.

S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Šresolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.

In silico discovery and validation of potent small-molecule inhibitors targeting the activation function 2 site of human oestrogen receptor α.

INTRODUCTION: Current approaches to inhibit oestrogen receptor-alpha (ERα) are focused on targeting its hormone-binding pocket and have limitations. Thus, we propose that inhibitors that bind to a coactivator-binding pocket on ERα, called activation function 2 (AF2), might overcome some of these limitations. METHODS: In silico virtual screening was used to identify small-molecule ERα AF2 inhibitors. These compounds were screened for inhibition of ERα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERα protein was measured using biolayer interferometry (BLI) and an ERα coactivator displacement assay. Cell viability was assessed by MTS assay in ERα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines TamR3 and TamR6, and ERα-negative MDA-MB-453 and HeLa cell lines. In addition, ERα inhibition in TamR cells and the effect of compounds on mRNA and protein expression of oestrogen-dependent genes, pS2, cathepsin D and cell division cycle 2 (CDC2) were determined. RESULTS: Fifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. In a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERα AF2 inhibitor that significantly downregulated ERα transcriptional activity (half-maximal inhibitory concentration = 5.81 µM). By directly binding to the ERα protein, as confirmed by BLI, VPC-16230 effectively displaced coactivator peptides from the AF2 pocket, confirming its site-specific action. VPC-16230 selectively suppressed the growth of ERα-positive breast cancer cells. Furthermore, it significantly inhibited ERα mediated transcription in TamR cells. More importantly, it reduced mRNA and protein levels of pS2, cathepsin D and CDC2, validating its ER-directed activity. CONCLUSION: We identified VPC-16230 as an ERα AF2-specific inhibitor that demonstrated promising antiproliferative effects in breast cancer cell lines, including TamR cells. VPC-16230 reduced the expression of ERα-inducible genes, including CDC2, which is involved in cell division. We anticipate that the application of ERα AF2 inhibitors will provide a novel approach that can act as a complementary therapeutic to treat ERα-positive, tamoxifen-resistant and metastatic breast cancers.

"From me to HIV": a case study of the community experience of donor transition of health programs.

BACKGROUND: Avahan, a large-scale HIV prevention program in India, transitioned over 130 intervention sites from donor funding and management to government ownership in three rounds. This paper examines the transition experience from the perspective of the communities targeted by these interventions. METHODS: Fifteen qualitative longitudinal case studies were conducted across all three rounds of transition, including 83 in-depth interviews and 45 focus group discussions. Data collection took place between 2010 and 2013 in four states: Andhra Pradesh, Karnataka, Maharashtra and Tamil Nadu. RESULTS: We find that communication about transition was difficult at first but improved over time, while issues related to employment of peer educators were challenging throughout the transition. Clinical services were shifted to government providers resulting in mixed experiences depending on the population being targeted. Lastly, the loss of activities aimed at community ownership and mobilization negatively affected the beneficiaries' view of transition. CONCLUSIONS: While some programmatic changes resulted in improvements, additional opportunity costs for beneficiaries may pose barriers to accessing HIV prevention services. Communicating and engaging community stakeholders early on in future such transitions may mitigate negative feelings and lead to more constructive relationships and dialogue.

Management practices to support donor transition: lessons from Avahan, the India AIDS Initiative.

BACKGROUND: During 2009-2012, Avahan, a large donor funded HIV/AIDS prevention program in India was transferred from donor support and operation to government. This transition of approximately 200 targeted interventions (TIs), occurred in three tranches in 2009, 2011 and 2012. This paper reports on the management practices pursued in support of a smooth transition of the program, and addresses the extent to which standard change management practices were employed, and were useful in supporting transition. RESULTS: We conducted structured surveys of a sample of 80 TIs from the 2011 and 2012 rounds of transition. One survey was administered directly before transition and the second survey 12 month after transition. These surveys assessed readiness for transition and practices post-transition. We also conducted 15 case studies of transitioning TIs from all three rounds, and re-visited 4 of these 1-3 years later. RESULTS: Considerable evolution in the nature of relationships between key actors was observed between transition rounds, moving from considerable mistrust and lack of collaboration in 2009 toward a shared vision of transition and mutually respectful relationships between Avahan and government in later transition rounds. Management practices also evolved with the gradual development of clear implementation plans, establishment of the post of "transition manager" at state and national levels, identified budgets to support transition, and a common minimum programme for transition. Staff engagement was important, and was carried out relatively effectively in later rounds. While the change management literature suggests short-term wins are important, this did not appear to be the case for Avahan, instead a difficult first round of transition seemed to signal the seriousness of intentions regarding transition. CONCLUSIONS: In the Avahan case a number of management practices supported a smooth transition these included: an extended and sequenced time frame for transition; co-ownership and planning of transition by both donor and government; detailed transition planning and close attention to program alignment, capacity development and communication; engagement of staff in the transition process; engagement of multiple stakeholders post transition to promote program accountability and provide financial support; signaling by actors in charge of transition that they were committed to specified time frames.

Comparison of Inflammatory Markers for Prediction of Recurrence of Nasal Polyp after Functional Endoscopy Sinus Surgery.

The study was carried out to correlate the inflammatory markers (NLR, ELR, PLR) before and after endoscopic sinus surgery and their role in the prediction of recurrent nasal polyps. This was a hospital-based observational study carried out the 43 patients, aged between 18-45 years, admitted to the department of ENT with CRSwNP and underwent endoscopic sinus surgery. NLR, ELR & PLR values were compared for each patient and calculated from complete blood counts taken before and after surgery follow-up period at post-op 1st week, 3rd week, 3rd month, and 6th month. In our study, 12 out of 43 patients who underwent ESS showed recurrence. The mean value of ELR was higher in the pre-operative and post-operative 1st week in recurrent nasal polyp patients than in non-recurrent nasal polyps. (p-value < 0.05) but there were no significant changes in ELR values in subsequent follow-ups. There were no significant changes in NLR & PLR in the pre-operative and post-operative periods. Although recurrence was common in CRSwNP after endoscopic sinus surgery. Inflammatory markers could be used to predict the chances of recurrence. In our study, ELR is a better parameter than NLR and PLR in the assessment of the chance of recurrence.

Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors.

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.

A comparison of intubating conditions for nasotracheal intubation with standard direct Macintosh laryngoscope versus C-MAC® video laryngoscope employing cuff inflation technique in adult patients.

BACKGROUND AND AIMS: While performing laryngoscopy during nasotracheal intubation (NTI), the tip of the advancing endotracheal tube (ETT) generally lies along the posterior pharyngeal wall. The inflation of the ETT cuff brings it anterior towards the glottis. The present study was planned to compare the intubating conditions for NTI with standard direct Macintosh laryngoscope versus C-MAC® video laryngoscope (VL) employing ETT cuff inflation technique. METHODS: This prospective randomised study was carried out on 50 patients, American Society of Anesthesiologists physical status I-II, age 18-60 years of either sex with an indication for NTI under general anaesthesia. They were randomly divided into two groups: group VL (n = 25): C-MAC® VL and group ML (n = 25): Macintosh laryngoscope. The primary outcome was to compare the total duration of NTI (T), while the secondary outcomes were to compare the need for cuff inflation or assistance with Magill forceps for successful NTI, the total number of attempts to achieve successful NTI, haemodynamic effects and complications. RESULTS: T was significantly higher in group ML than group VL (P < 0.001). The intubation was successful with cuff inflation in all the patients in group VL, however, six patients of group ML required assistance with Magill forceps (P = 0.022). The haemodynamic parameters were all significantly higher at 3 min in group ML in comparison to group VL. CONCLUSION: The cuff inflation technique when used along with C-MAC® VL had more success rate, required lesser time and had minimal postoperative complications in comparison to the Macintosh laryngoscope.

Intra-Clade C signature polymorphisms in HIV-1 LTR region: The Indian and African lookout.

Polymorphisms occurring in LTR (Long Terminal Repeat) region can profoundly impact pathogenicity, transmission and biology of Human Immunodeficiency Virus Type 1 (HIV-1). We investigated intra-clade polymorphisms, associated with HIV-1 clade-C infections that occur in India and Africa. Plasma samples were obtained from 24 HIV-infected ART-experienced individuals. Next Generation Sequencing was performed on Illumina Hi Seq X system. Sequence analysis was done using MEGA v7. Transcription factor binding sites (TFBS) were investigated to unveil signature sequences. Signature nucleotides in Indian sequences were observed at 19 positions, of which 7 nucleotide signatures occurred in transcription binding sites (TFBS), namely NF-AT-II, NF-AT-III, USF, TCF- 1alpha, Sp1-I and TAR. Intra-clade C variations in HIV-1 LTR that inscribe signature nucleotides in Indian sequences lead to formation monophyletic cluster of Indian sequences. Moreover, occurrence of intra-clade signature nucleotides was observed at the key positions in the transcription factor binding sites in Indian and African clade-C sequences.

Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer.

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

Androgen receptor plasticity and its implications for prostate cancer therapy.

Acquired resistance to a drug treatment is a common problem across many cancers including prostate cancer (PCa) - one of the major factors for male mortality. The androgen receptor (AR) continues to be the main therapeutic PCa target and despite the success of modern targeted therapies such as enzalutamide, resistance to these drugs eventually develops. The AR has found many ways to adapt to treatments including overexpression and production of functional, constitutively active splice variants. However, of particular importance are point mutations in the ligand binding domain of the protein that convert anti-androgens into potent AR agonists. This mechanism appears to be especially prevalent with the AR in spite of some distant similarities to other hormone nuclear receptors. Despite the AR being one of the most studied and attended targets in cancer, those gain-of-function mutations in the receptor remain a significant challenge for the development of PCa therapies. This drives the need to fully characterize such mutations and to consistently screen PCa patients for their occurrence to prevent adverse reactions to anti-androgen drugs. Novel treatments should also be developed to overcome this resistance mechanism and more attention should be given to the possibility of similar occurrences in other cancers.

Comparative evaluation of Ambu AuraGain™ with ProSeal™ laryngeal mask airway in patients undergoing laparoscopic cholecystectomy.

BACKGROUND AND AIMS: Second generation supraglottic airways are increasingly being used in surgical patients undergoing laparoscopic surgery. Preventing aspiration at higher airway pressures may be at the expense of a higher cuff pressure which can impair mucosal perfusion. We attempted to elucidate whether Ambu AuraGain™ (AAU) would provide a higher oropharyngeal leak pressure (OLP) with a lower mucosal pressure in comparison to ProSeal™ laryngeal mask airway (PLMA). METHODS: This was a prospective randomised study involving sixty patients undergoing laparoscopic cholecystectomy under general anaesthesia, using either AAU (Group AAU [n = 30]) or PLMA (Group PLMA [n = 30]) for elective ventilation. Primary outcome measure was the OLP. Number of insertion attempts, ease of insertion, time required for placement and calculated pharyngeal mucosal pressure were the secondary outcome measures. Data were analysed using Student's t-test and Chi-square test. RESULTS: No significant difference in the OLP was noted in both groups. The ease of insertion and success rate at first attempt was similar between the groups. Time taken for insertion in Group AAU was longer than Group PLMA (13.57 ± 1.94 vs. 11.60 ± 2.22 s). The calculated pharyngeal mucosal pressures were lower with Group AAU than Group PLMA for all 3 sizes. The minimum cuff pressure and minimum cuff volume required to prevent leak were found similar in both groups. CONCLUSION: AAU provides adequate sealing pressures and effective ventilation with lower calculated pharyngeal mucosal pressure, compared to PLMA.

The miR-200 family is increased in dysplastic lesions in ulcerative colitis patients.

BACKGROUND: Colorectal cancer (CRC) is a life-threatening complication of ulcerative colitis (UC), and patients are routinely screened for the development of precancerous lesions (dysplasia). However, rates of CRC development in patients with confirmed low-grade dysplasia vary widely between studies, suggesting a large degree of heterogeneity between these lesions that is not detectable macroscopically. A better understanding of the underlying molecular changes that occur in dysplasia will help to identify lesions at higher risk of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate protein expression and cell-signalling networks. Aberrant miRNA expression is a feature of sporadic CRC but much less is known about the changes that occur in dysplasia and in UC. METHODS: Comprehensive microRNA profiling was performed on RNA extracted from UC dysplastic lesions (n = 7) and UC controls (n = 10). The expression of miRNAs in UC post inflammatory polyps (n = 7) was also assessed. Candidate miRNAs were further validated by qPCR, and miRNA in situ hybridization. Serum levels of miRNAs were also assessed with a view to identification of non-invasive biomarkers of dysplasia. RESULTS: UC dysplasia was associated with a shift in miRNA expression profiles that was not seen in inflammatory polyps. In particular, levels of miR-200b-3p were increased in dysplasia, and this miRNA was localised to epithelial cells in dysplastic lesions and in UC cancers. No changes in miRNA levels were detected in the serum. CONCLUSION: UC-Dysplasia is linked to altered miRNA expression in the mucosa and elevated miR-200b-3p levels.