Future Drug Development Strategies Research Articles

Stability-based approaches in chemoproteomics.

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Nyctanthes arbor-tristis bioactive extract ameliorates LPS-induced inflammation through the inhibition of NF-κB signalling pathway

Ethnopharmacological relevanceNyctanthes arbor-tristis L. is a mythical plant used in traditional Indian medicinal systems for the treatment of inflammation, rheumatoid arthritis, and pain-related responses. However, its bioactive compounds and underlying mechanism of action have not been fully elucidated. Aim of the studyThis investigation aimed to study the anti-inflammatory and anti-nociceptive effects of the bioactive extract of N. arbor-tristis (NATE), both in vitro and in vivo, elucidate the possible mechanism of action, and determine its chemicals. Materials and methodsWe studied the anti-inflammatory and anti-nociceptive activities of NATE on lipopolysaccharide-stimulated RAW264.7 macrophages, paw-ear edema, and acetic acid-induced pain in rats and analysed its chemical components using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS). ResultsNATE efficiently reduced the production of various inflammatory mediators and factors, such as free radicals, lipid peroxidation, nitrous oxide (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-1beta (IL-1β), and IL-10, as well as their corresponding mRNA expression in LPS-induced RAW264.7 cells (p < 0.001). Furthermore, NATE inhibited the activation of a key signaling pathway, nuclear factor-kappa B (NF-kB), as it caused a decrease in the degradation of inhibitor of kB alpha (IkBa). Administration of NATE significantly inhibited carrageenan-induced paw edema (p < 0.001), TPA-induced ear edema, and the production of inflammatory factors (p < 0.01). NATE revealed anti-nociceptive impacts in acetic acid-induced writhing and tail immersion experiments (p < 0.001) as well as no toxicity signs. A total of six compounds, namely iridoid glycoside (6,7-di-O-benzonylnyctanthoside), arborsides A, arborsides C, betulinic acid, kaempferol 3-O-glucoside, and kaempferol 3-O-rutinoside, were characterized through the examination of their mass spectra in correlation with those documented in a database of mass spectra. ConclusionsThe present study furnishes scientific corroboration of the inhibitory potency of N. arbor-tristis as a promising herbal treatment for inflammation and pain responses without toxicity, offering a scientific basis for future drug development strategies aimed at ameliorating inflammatory diseases.

Current and future therapeutic strategies for Alzheimer's disease: an overview of drug development bottlenecks.

Alzheimer's disease (AD) is the most common chronic neurodegenerative disease worldwide. It causes cognitive dysfunction, such as aphasia and agnosia, and mental symptoms, such as behavioral abnormalities; all of which place a significant psychological and economic burden on the patients' families. No specific drugs are currently available for the treatment of AD, and the current drugs for AD only delay disease onset and progression. The pathophysiological basis of AD involves abnormal deposition of beta-amyloid protein (Aβ), abnormal tau protein phosphorylation, decreased activity of acetylcholine content, glutamate toxicity, autophagy, inflammatory reactions, mitochondria-targeting, and multi-targets. The US Food and Drug Administration (FDA) has approved five drugs for clinical use: tacrine, donepezil, carbalatine, galantamine, memantine, and lecanemab. We have focused on the newer drugs that have undergone clinical trials, most of which have not been successful as a result of excessive clinical side effects or poor efficacy. Although aducanumab received rapid approval from the FDA on 7 June 2021, its long-term safety and tolerability require further monitoring and confirmation. In this literature review, we aimed to explore the possible pathophysiological mechanisms underlying the occurrence and development of AD. We focused on anti-Aβ and anti-tau drugs, mitochondria-targeting and multi-targets, commercially available drugs, bottlenecks encountered in drug development, and the possible targets and therapeutic strategies for future drug development. We hope to present new concepts and methods for future drug therapies for AD.

Evaluation of Saxifraga stolonifera phenolic extracts as a potential antivenom against Deinagkistrodon acutus venom: In vitro and in vivo studies

In the snake-infested mountainous regions of China, Saxifraga stolonifera [L.] Meeb is widely utilized as an immediate remedy for venomous snake bites. However, the scientific understanding of S. stolonifera's efficacy in snakebite treatment remains limited and requires further investigation. The aim of this study was to assess the inhibitory effects of Saxifraga stolonifera phenolic extracts (SSPE) on Deinagkistrodon acutus venom (DAV) and explore the potential of S. stolonifera as a valuable candidate for antivenom development. We employed our previously optimized extraction conditions to obtain SSPE. In vitro experiments utilizing diverse models were conducted to assess the inhibitory effects of the extracted phenolic compounds on DAV, specifically targeting phospholipase A2 (PLA2), proteolytic, fibrinolytic, and hyaluronidase enzymes. Furthermore, in vivo investigations were conducted to evaluate the inhibitory potential of the extracted compounds against DAV-induced hemorrhagic and edematogenic activity. To elucidate the chemical composition of the phenolic extracts, Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS) analysis was performed. Our in vitro inhibition study showed that S. stolonifera was able to inhibit the activities of PLA2 enzyme, proteolytic enzyme, hyaluronidase and fibrinogenolytic. The median effective dose (ED50) values of SSPE for inhibiting PLA2 enzyme, proteolytic enzyme and hyaluronidase activities were 0.115 mg/mL, 0.026 mg/mL and 0.238 mg/mL, respectively. The DAV-induced hemorrhagic and edematogenic effects of the SSPE were also successfully inhibited in vivo, and the high SSPE concentration was able to completely inhibit the hemorrhage and edema. It is noteworthy that the mice suffered no harm from the high SSPE concentration. The composition analysis showed that the phenolic substances contained in SSPE are gallic acid, protocatechuic acid, chlorogenic acid, rutin, kaempferol-3-O-ɑ-L-rhamnoside, kaempferol-3-O-β-D-glucopyranoside, quercetin and kaempferol. This study provides scientific validation of the inhibitory efficacy of S. stolonifera as an emergency treatment for venomous snake bites, offering a theoretical foundation for future drug development strategies targeting snakebite.

Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging.

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

Evolution of Phase II Oncology Trial Design: from Single Arm to Master Protocol.

The recent development of novel anticancer treatments with diverse mechanisms of action has accelerated the detection of treatment candidates tremendously. The rapidly changing drug development landscapes and the high failure rates in Phase III trials both underscore the importance of more efficient and robust phase II designs. The goals of phase II oncology studies are to explore the preliminary efficacy and toxicity of the investigational product and to inform future drug development strategies such as go/no-go decisions for phase III development, or dose/indication selection. These complex purposes of phase II oncology designs call for efficient, flexible, and easy-to-implement clinical trial designs. Therefore, innovative adaptive study designs with the potential of improving the efficiency of the study, protecting patients, and improving the quality of information gained from trials have been commonly used in Phase II oncology studies. Although the value of adaptive clinical trial methods in early phase drug development is generally well accepted, there is no comprehensive review and guidance on adaptive design methods and their best practice for phase II oncology trials. In this paper, we review the recent development and evolution of phase II oncology design, including frequentist multistage design, Bayesian continuous monitoring, master protocol design, and innovative design methods for randomized phase II studies. The practical considerations and the implementation of these complex design methods are also discussed.

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in glioblastoma.

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

Multipronged diagnostic and therapeutic strategies for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and a major contributor to dementia cases worldwide. AD is clinically characterized by learning, memory, and cognitive deficits. The accumulation of extracellular amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs) of tau are the pathological hallmarks of AD and are explored as targets for clinical diagnosis and therapy. AD pathology is poorly understood and there are no fully approved diagnosis and treatments. Notwithstanding the gap, decades of research in understanding disease mechanisms have revealed the multifactorial nature of AD. As a result, multipronged and holistic approaches are pertinent to targeting multiple biomarkers and targets for developing effective diagnosis and therapeutics. In this perspective, recent developments in Aβ and tau targeted diagnostic and therapeutic tools are discussed. Novel indirect, combination, and circulating biomarkers as potential diagnostic targets are highlighted. We underline the importance of multiplexing and multimodal detection of multiple biomarkers to generate biomarker fingerprints as a reliable diagnostic strategy. The classical therapeutics targeting Aβ and tau aggregation pathways are described with bottlenecks in the strategy. Drug discovery efforts targeting multifaceted toxicity involving protein aggregation, metal toxicity, oxidative stress, mitochondrial damage, and neuroinflammation are highlighted. Recent efforts focused on multipronged strategies to rationally design multifunctional modulators targeting multiple pathological factors are presented as future drug development strategies to discover potential therapeutics for AD.

Host DNA repair response to oxidative damage is modulated by Trypanosoma cruzi in a strain-dependent manner

The conservation of genomic integrity and stability is essential for cell survival. DNA Damage Responses (DDRs) are considered of paramount importance for all living beings and involve mechanisms of cell cycle regulation and damage-specific DNA repair pathways. Hydrogen peroxide (H2O2) is a compound that, in supraphysiological concentrations, damages biomolecules including the DNA, causing base modifications and strand breaks. There is evidence that Trypanosoma cruzi, the protozoan that causes Chagas disease, interferes in the host cell's DNA metabolism. In order to investigate the influence of T. cruzi infection over the host cell capacity to withstand and repair DNA damage, we analyzed L6 cells infected with Berenice, and Colombiana T. cruzi strains according to their viability, proliferation, morphology, DNA degradation, expression of DNA repair, and cell cycle genes following H2O2 treatment. It was noted that T. cruzi infection might act as either a stressor or a protective element of host DNA, depending on the strain and H2O2 concentration. Cells infected with Berenice strain and treated with 0.8 mM H2O2 presented a reduced DNA damage response intensity (e.g., BER and HR). Infection with T. cruzi Colombiana prevented the activation of DNA repair pathways in response to 0.8mM and 1.6mM H2O2 (NER and MMR). Nevertheless, since cellular viability was not significantly compromised in Colombiana-infected cells following the oxidative insult, it is possible that the parasite directly influenced the host DNA repair machinery. Our results support the notion that T. cruzi is able to modulate the host cell DNA metabolism in a strain-dependent manner, an event which can be explored in future drug development strategies.

Mice lacking melatonin MT2 receptors exhibit attentional deficits, anxiety and enhanced social interaction.

Background:Aside from regulating circadian rhythms, melatonin also affects cognitive processes, such as alertness, and modulates the brain circuitry underlying psychiatric diseases, such as depression, schizophrenia and bipolar disorder, via mechanisms that are not fully clear. In particular, while melatonin MT1 receptors are thought primarily to mediate the circadian effects of the hormone, the contribution of the MT2 receptor to melatonin actions remains enigmatic.Aims:To characterise the contribution of MT2 receptors to melatonin’s effects on cognition and anxiety/sociability.Methods:Mice with a genetic deletion of the MT2 receptor, encoded by the Mtnr1b gene, were compared with wild-type littermates for performance in a translational touchscreen version of the continuous performance task (CPT) to assess attentional processes and then monitored over 3 days in an ethological home-cage surveillance system.Results:Mtnr1b knockout (KO) mice were able to perform at relatively normal levels in the CPT. However, they showed consistent evidence of more liberal/risky responding strategies relative to control mice, with increases in hit rates and false alarm rates, which were maintained even when the cognitive demands of the task were increased. Assessment in the home-cage monitoring system revealed that female Mtnr1b KO mice have increased anxiety levels, whereas male Mtnr1b KO mice show increased sociability.Conclusions:The results confirm that the MT2 receptor plays a role in cognition and also modulates anxiety and social interactions. These data provide new insights into the functions of endogenous melatonin and will inform future drug development strategies focussed on the MT2 receptor.

Immune-Checkpoint Inhibitors in Platinum-Resistant Ovarian Cancer

Simple SummaryPatients with platinum-resistant ovarian cancer experience poor prognosis. No mature evidence supports the routine adoption of immunotherapy alone in this setting. However, the combination of immunotherapy with target therapies seems to be a promising option in patients with ovarian cancer. Ongoing trials are testing the combination between immune therapy and other target therapies, including PARP inhibitors, TKI, and anti-angiogenetic therapies. Further evidence is needed to assess the real impact and cost-effectiveness of immmunotherapic agents in platinum-resistant ovarian cancer.Platinum-resistant ovarian cancer (OC) has limited treatment options and is associated with a poor prognosis. There appears to be an overlap between molecular mechanisms responsible for platinum resistance and immunogenicity in OC. Immunotherapy with single agent checkpoint inhibitors has been evaluated in a few clinical trials with disappointing results. This has prompted exploration of immunotherapy combination strategies with chemotherapy, anti-angiogenics, poly (ADP-ribose) polymerase (PARP) inhibitors and other targeted agents. The role of immunotherapy in the treatment of platinum-resistant OC remains undefined. The aim of this review is to describe the immunobiology of OC and likely benefit from immunotherapy, discuss clinical trial data and biomarkers that warrant further exploration, as well as provide an overview of future drug development strategies.

Evaluation of the alpha-amylase inhibitory activity of Nepalese medicinal plants used in the treatment of diabetes mellitus

Backgroundα-Amylase catalyses the hydrolysis of starch and ultimately producing glucose. Controlling the catalytic activity of this enzyme reduces glucose production in the postprandial stage, which could be a therapeutic benefit for people with diabetes. This study was conducted to evaluate α-amylase inhibition for utilizing the crude extracts of some medicinal plants traditionally used in Nepal for the treatment of diabetes and its related complications.MethodsMicrotiter plate approach has been used to assess inhibitory activities of in vitro α-amylase of methanolic extracts of thirty-two medicinal plants. A starch tolerance test was used in rats to investigate the in vivo study of the methanolic extract concerning glibenclamide as the positive control.ResultsAcacia catechu, Dioscorea bulbifera, and Swertia chirata exhibited inhibitory activity against α-amylase and with IC50 values; 49.9, 296.1, and 413.5 μg/mL, respectively. Kinetics study revealed that all the extracts displayed a mixed type of inhibition pattern, with Ki values ranging from 26.6–204.2 μg/mL. Free radical scavenging activity was again re-examined and found prominent in extracts of A. catechu. Likewise, A. catechu and S. chirata showed significant reduction of blood glucose concentration up to 30 min after oral dose of 250 mg/kg (F (4, 20) = 4.1, p = .048), and (F (4, 20) = 4.1, p = .036), respectively.ConclusionsEnzymatic assay for α-amylase inhibition using extracts was successfully evaluated. Also, the in-vitro and in-vivo study model revealed that medicinal plants could be a potent source of α-amylase inhibition. So, they could serve as potential candidates for future drug development strategies for curing diabetes with minimal or no adverse side effects.

Plasmodium falciparum Artemisinin Resistance: The Effect of Heme, Protein Damage, and Parasite Cell Stress Response.

Despite a significant decline in morbidity and mortality over the last two decades, in 2018 there were 228 million reported cases of malaria and 405000 malaria-related deaths. Artemisinin, the cornerstone of artemisinin-based combination therapies, is the most potent drug in the antimalarial armamentarium against falciparum malaria. Heme-mediated activation of artemisinin and its derivatives results in widespread parasite protein alkylation, which is thought to lead to parasite death. Alarmingly, cases of decreased artemisinin efficacy have been widely detected across Cambodia and in neighboring countries, and a few cases have been reported in the Guiana Shield, India, and Africa. The grim prospect of widespread artemisinin resistance propelled a concerted effort to understand the mechanisms of artemisinin action and resistance. The identification of genetic markers and the knowledge of molecular mechanisms underpinning artemisinin resistance allow prospective surveillance and inform future drug development strategies, respectively. Here, we highlight recent advances in our understanding of how parasite vesicle trafficking, hemoglobin digestion, and cell stress responses contribute to artemisinin resistance.

Conformational Landscapes of HER2 Exon 20 Insertions Explain Their Sensitivity to Kinase Inhibitors in Lung Adenocarcinoma.

HER2 exon 20 insertion (ex20ins) is one of the most intractable problems in lung cancer. Most ex20ins are resistant to available EGFR or pan-HER tyrosine kinase inhibitors (TKIs), with the exception of a few mutants. However, the mechanism for TKI response and resistance of HER2 ex20ins remains poorly understood. Next-generation sequencing-based genomic profiling data of 4139 patients with lung cancer were interrogated for HER2 ex20ins. Structural modeling and molecular dynamics simulations of common HER2 ex20ins were carried out to provide insights into the mechanism of activation and response heterogeneity of ex20ins. Molecular docking was performed to predict affinity to TKIs. Therapeutic decisions for patients were made on the basis of the results of genomic profiling. From 155 HER2-mutant lung cancer cases, Y772_A775dup and G778_P780dup were identified in 74 (47.7%) and 18 (11.6%) cases, respectively. Molecular dynamics simulations revealed that HER2 ex20ins led to ligand-independent kinase activation by changing the conformational landscape of HER2 kinase and restricting kinase conformation in the active state. G778_P780dup had a three-amino acid extension in the αC-β4 loop and retained the HER2-characteristic G776 and G778. Compared with Y772_A775dup, it had less restriction on kinase conformational sampling and higher affinity to afatinib, dacomitinib, pyrotinib, and poziotinib. Treating lung adenocarcinomas carrying G778_P780dup with these inhibitors led to sustained tumor responses in six of the 10 patients. The kinase conformational landscape dictated by the length of the αC-β4 loop and residues at HER2 776 and 778 position explains TKI sensitivity in ex20ins. This finding could guide therapeutic decisions with currently available therapies and future drug development strategies.

The effects of the DNA Demethylating reagent, 5-azacytidine on SMCHD1 genomic localization

BackgroundDNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC).ResultsOur data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression.ConclusionThese findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

The endocannabinoid system: from the receptors to therapy

Abstract The endocannabinoid system (ECS) received a lot of attention ever since its discovery. Advancements of the last three decades have shown that there are numerous mechanisms by which the ECS regulates the energy metabolism. These can either be central (regulating appetite and calorie expenditure) or peripheral (adipocyte-specific and other) mechanisms. The current review highlights some of the most important observations leading to the discovery of the ECS first, followed by a part detailing the synthesis and transport of these mediators, the receptor types and second messenger systems involved. The next part is dedicated to the mechanisms by which this system regulates the energy metabolism. Lastly, the drugs that reached the clinical phase and the main targets and strategies for future drug development will be reviewed.

SUN-005 DAX-1 Inhibition of Estrogen Receptor and Metastasis Target Genes in Estrogen Receptor Positive Breast Cancer Cells

The association of estrogens with breast cancer has led to the development of drugs that block the action of estrogens to treat and prevent estrogen receptor (ER) positive tumors. However, many tumors eventually become resistant to these drugs. Future drug discovery requires the identification of novel targets in the ER signaling pathway. The identification of proteins that directly bind to ER may offer new therapeutic targets for the treatment of breast cancer. The orphan receptor, DAX-1 (Dosage Sensitive Sex Reversal Adrenal Hypoplasia Congenita on the X Chromosome, gene 1) has been shown to act as a corepressor of ERα actions in MCF7 breast cancer cells. In this study we investigated the expression of DAX-1 during progressive stages of invasive ductal carcinoma with known ER status. To define the effect of DAX-1 expression in ER positive breast cancer cells, we performed gene expression analysis using PCR arrays that were specific for human breast cancer gene targets, human estrogen receptor signaling targets and tumor metastasis targets. We found that adenovirus-mediated expression of DAX-1 inhibits MCF7 cell proliferation and blocks estradiol activation of specific ER target genes, including cyclin D1. Repression of the cyclin D1 gene in MCF7 cells is associated with the recruitment of DAX-1 to the cyclin D1 promoter. Furthermore, introduction of DAX-1 into MCF7 cells leads to cell cycle arrest and an increase in apoptosis. These results demonstrate that DAX-1 functions as a corepressor of ER action in MCF7 breast cancer cells by preventing the activation of growth promoting genes and decreasing the stimulation of cell proliferation in response to estrogens. These finding could have significant implications for future drug development strategies specifically aimed at ER interacting proteins such as DAX-1.

Abstract 2533: Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells

Abstract The orphan nuclear receptor DAX-1 (Dosage Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, critical region on the X chromosome, gene 1) is known to serve an important role early on in mammalian sex determination as well as in the regulation of expression of steroid hormones. While it is unclear whether DAX-1 functions independently, DAX-1 has been demonstrated to act as a repressor of many nuclear hormone receptors, including ER, AR, PR, LRH-1 and SF-1. DAX-1 plays a role in select adult tissues, including gonadal tissue. However, it has also been shown to play a role in several types of cancer, though its specific function is not well understood. For example, in some types of cancer DAX-1 expression is upregulated, while in other cancers it is highly downregulated or completely absent. In an effort to better understand DAX-1 function both in normal and disease states, we examined the actions of DAX-1 in MCF7 breast cancer cells, which do not express the DAX-1 gene. We probed three different PCR arrays (breast cancer genes, proliferation genes and metastatic genes) to determine which genes were influenced by DAX-1 expression. With the introduction of DAX-1 in ERα positive breast cancer cells (MCF-7), we find that DAX-1 inhibits cell proliferation and blocks estradiol activation of key cell cycle regulatory genes, such as cyclin D1, as well as important metastatic genes. We show that DAX-1 binds directly to ERα and the ERα-DAX-1 complex binds to the cyclin D1 promoter. The outcome of DAX-1 expression in MCF7 cells is cell cycle arrest through the repression of key cell cycle regulatory genes. These results demonstrate that DAX-1 functions as a corepressor of ERα in MCF7 breast cancer cells by preventing the activation of growth-promoting genes and decreasing cell proliferation in response to estrogens. These findings could have significant implications for future drug development strategies specifically aimed at ER-interacting proteins such as DAX-1. Citation Format: Christina Tzagarakis-Foster. Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2533.

Proteomic Profile of Circulating Inflammatory Proteins Associated with 10-Year Risk of ESRD in T1 and T2 Diabetes—Enrichment for TNF Receptor Superfamily Members

We conducted an untargeted proteomic profiling of circulating inflammatory proteins using SOMASCAN platform to find those that predicted 10-year ESRD risk in two cohort study. Subjects with Type 1 Diabetes (T1D) served as a discovery cohort and a T2D cohort was used in validation. We identified a kidney risk inflammatory signature (KRIS) that robustly predicted progression to ESRD. The signature comprised 17 proteins out of 194 examined. KRIS was enriched for members of TNFR Superfamily (p=0.007) including TNFR1, TNFR2, TNFRSF19, TNFRSF19L, TNFRSF21 and TNFRSF27. Other KRIS proteins included IL15RA, IL17F, DAF, CLM6, TNFSF15, CCL14, CCL15, CSF1, TIMD3, IL1R1 and IL18R1. Hazard ratio (95% CI) for the top KRIS protein, TNFR1 was 3.6 (2.8, 4.6), p &amp;lt; 10-25. KRIS was neither enriched in receptors for other cytokines nor in cytokine ligands. Pathway analyses pointed to candidate therapeutic targets and aligned them by ranks. Eight proteins are currently targeted with compounds used for other clinical indications. Kidney tissue expression analysis and urinary proteomics suggested a systemic source of KRIS proteins. In summary, our study identifies and validates a powerful protein signature enriched in TNFRSF members associated with 10-year ESRD risk in diabetes. These findings should inform future drug development strategies. Disclosure M. Niewczas: None. J.K. Skupien: None. V. Nair: None. A. Smiles: None. J. Park: None. E. Satake: None. C.A. Simeone: None. J. Tsay: None. W. Ju: None. M. Kretzler: Research Support; Self; AstraZeneca, Novo Nordisk Inc., Boehringer Ingelheim GmbH, National Institute of Diabetes and Digestive and Kidney Diseases, Eli Lilly and Company, Gilead Sciences, Inc. K. Susztak: Research Support; Self; Regeneron Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., GlaxoSmithKline plc., Merck &amp; Co., Inc., Eli Lilly and Company, Ono Pharmaceutical Co., Ltd.. A. Krolewski: None.