Structure-based discovery of a novel small-molecule inhibitor of TEAD palmitoylation with anticancer activity.

The paralogous oncogenic transcriptional coactivators YAP and TAZ are the distal effectors of the Hippo signaling pathway, which plays a critical role in cell proliferation, survival and cell fate specification. They are frequently deregulated in most human cancers, where they contribute to multiple aspects of tumorigenesis including growth, metabolism, metastasis and chemo/immunotherapy resistance. Thus, they provide a critical point for therapeutic intervention. However, due to their intrinsically disordered structure, they are challenging to target directly. Since YAP/TAZ exerts oncogenic activity by associating with the TEAD1-4 transcription factors, to regulate target gene expression, YAP activity can be controlled indirectly by regulating TEAD1-4. Interestingly, TEADs undergo autopalmitoylation, which is essential for their stability and function, and small-molecule inhibitors that prevent this posttranslational modification can render them unstable. In this article we report discovery of a novel small molecule inhibitor of YAP activity. We combined structure-based virtual ligand screening with biochemical and cell biological studies and identified JM7, which inhibits YAP transcriptional reporter activity with an IC50 of 972 nMoles/Ltr. Further, it inhibits YAP target gene expression, without affecting YAP/TEAD localization. Mechanistically, JM7 inhibits TEAD palmitoylation and renders them unstable. Cellular thermal shift assay revealed that JM7 directly binds to TEAD1-4 in cells. Consistent with the inhibitory effect of JM7 on YAP activity, it significantly impairs proliferation, colony-formation and migration of mesothelioma (NCI-H226), breast (MDA-MB-231) and ovarian (OVCAR-8) cancer cells that exhibit increased YAP activity. Collectively, these results establish JM7 as a novel lead compound for development of more potent inhibitors of TEAD palmitoylation for treating cancer.

Organometallic Folate Gold Nanoparticles Ameliorate Lipopolysaccharide-Induced Oxidative Damage and Inflammation in Zebrafish Brain.

Synthesized organometallic gold-based folate nanoparticles (FAuNPs) were characterized, and its defense against lipopolysaccharide (LPS)-induced brain inflammation in Zebra fish was proven. Vitamin entrapment efficiency of these particles was found to be nearly 70%. The in vitro pH-dependent drug release dialysis study of FAuNPs confirmed a slow, sustained, and gradual release of folate for a period of 24 h. Both AuNPs and FAuNPs did not cause any marked changes in food intake, body weight, color, behavioral pattern, blood parameters, and hepatotoxicity. Histology of liver showed no changes between treated and control groups of fishes. The ex vivo study showed significant uptake of FAuNPs to free folate in folate receptor negative Hek293 cells, confirming a strategy to overcome folate deficiency in the brain. Antioxidant status and activities of few crucial brain enzymes were also measured to assess the brain function and found to be returned to the basal level, following FAuNP treatment. The transcription factor NRF2-Keap 1 expression pattern was also noted, and a prominent modulation was observed in the LPS-treated and FAuNP-administered group. Decisive brain enzymes like AChE and Na+K+ATPase were decreased significantly after LPS treatment, which is restored with FAuNP treatment. Caspases increased sharply after LPS treatment and diminished following FAuNP treatment. We conclude that FAuNP due to its high physical stability and uptake could be utilized against severe brain inflammation, leading to brain injury and neurodegeneration.

Assays Used for Discovering Small Molecule Inhibitors of YAP Activity in Cancers.

YAP/TAZ are transcriptional coactivators that function as the key downstream effectors of Hippo signaling. They are commonly misregulated in most human cancers, which exhibit a higher level of expression and nuclear localization of YAP/TAZ, and display addiction to YAP-dependent transcription. In the nucleus, these coactivators associate with TEA domain transcription factors (TEAD1-4) to regulate the expression of genes that promote cell proliferation and inhibit cell death. Together, this results in an excessive growth of the cancerous tissue. Further, YAP/TAZ play a critical role in tumor metastasis and chemotherapy resistance by promoting cancer stem cell fate. Furthermore, they affect tumor immunity by promoting the expression of PD-L1. Thus, YAP plays an important role in multiple aspects of cancer biology and thus, provides a critical target for cancer therapy. Here we discuss various assays that are used for conducting high-throughput screens of small molecule libraries for hit identification, and subsequent hit validation for successful discovery of potent inhibitors of YAP-transcriptional activity. Furthermore, we describe the advantages and limitations of these assays.

Molecular Insight into the Effect of a Single-Nucleotide Polymorphic Variation on the Structure and Dynamics of Methionine Synthase Reductase and Its Association with Neural Tube Defects.

Neural tube defects (NTDs) are among the common and severe congenital malformations in neonates. According to a WHO report, nearly three lakh babies are affected per year worldwide by NTDs. Most studies revealed that folate deficiency is the key element to promote NTD with other oligogenic and multifactorial elements. This folate is metabolized by the FOCM (folate one-carbon metabolism) pathway. The most important step in the FOCM pathway is the conversion of methionine to homocysteine, which is guided by the enzyme MTRR. Several single-nucleotide polymorphisms (SNPs) in the MTRR gene are strongly associated with the progression of NTD. A nonsynonymous allelic variant (rs1532268) of the protein leads to a missense mutation at the 202nd position from serine to leucine (S202L) and is associated with a higher disease prevalence in different populations. In our study, this SNP indicates a 2-fold increase in the risk of disease progression (p-value of 0.03; OR 2.76; 95% CI 1.08-7.11). Here, extensive molecular dynamics simulations and interaction network analysis reveal that the change of 202nd serine to leucine alters the structures of the FAD and NAD binding domains, which restricts the ligand binding. The S202L variation alters the functional dynamics that might impede the electron transport chain along the NADP(H)→ FAD→ FMN pathway and hamper phosphorylation by kinases like GSK-3 and CaM-II during the posttranscriptional modification of the protein. The present study provides functional insights into the effect of the genetic variations of the MTRR gene on the NTD disease pathogenesis.

Therapeutic Potential of Exploiting Autophagy Cascade Against Coronavirus Infection.

Since its emergence in December 2019 in Wuhan, China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created a worldwide pandemic of coronavirus disease (COVID-19) with nearly 136 million cases and approximately 3 million deaths. Recent studies indicate that like other coronaviruses, SARS-CoV-2 also hijacks or usurps various host cell machineries including autophagy for its replication and disease pathogenesis. Double membrane vesicles generated during initiation of autophagy cascade act as a scaffold for the assembly of viral replication complexes and facilitate RNA synthesis. The use of autophagy inhibitors - chloroquine and hydroxychloroquine initially appeared to be as a potential treatment strategy of COVID-19 patients but later remained at the center of debate due to high cytotoxic effects. In the absence of a specific drug or vaccine, there is an urgent need for a safe, potent as well as affordable drug to control the disease spread. Given the intricate connection between autophagy machinery and viral pathogenesis, the question arises whether targeting autophagy pathway might show a path to fight against SARS-CoV-2 infection. In this review we will discuss about our current knowledge linking autophagy to coronaviruses and how that is being utilized to repurpose autophagy modulators as potential COVID-19 treatment.

Structural insight into the effect of polymorphic variation on the functional dynamics of methionine synthase reductase: Implications in neural tube defects.

Neural tube defects (NTDs), one of the most common birth defects, are strongly associated with the variations of several single nucleotide polymorphisms (SNPs) in the MTRR gene. The gene codes a key enzyme that is involved in the rejuvenation of methionine synthase activity. An allelic variant of the protein leads to missense mutation at 49th position from isoleucine to methionine (I49M) is associated with higher disease prevalence in different populations. Here, extensive molecular dynamics simulations and interaction network analysis reveal that the 49th isoleucine is a crucial residue that allosterically regulates the dynamics between the flavin mononucleotide (FMN) and NADP(H) binding domains. I49M variation alters the functional dynamics in a way that might impede the electron transport chain along the NADP(H) â†’ flavin adenine dinucleotide â†’ FMN pathway. The present study provides functional insights into the effect of the genetic variations of the MTRR gene on the NTDs disease pathogenesis.

Preparation, characterization and in vivo evaluation of pH sensitive, safe quercetin-succinylated chitosan-alginate core-shell-corona nanoparticle for diabetes treatment.

The study aims for development of an efficient polymeric carrier for evaluating pharmaceutical potentialities in modulating the drug profile of quercetin (QUE) in anti-diabetic research. Alginate and succinyl chitosan are focused in this investigation for encapsulating quercetin into core-shell nanoparticles through ionic cross linking. The FT-IR, XRD, NMR, SEM, TEM, drug entrapment and loading efficiency are commenced to examine the efficacy of the prepared nanoparticles in successful quercetin delivery. Obtained results showed the minimum particle size of ∼91.58nm and ∼95% quercetin encapsulation efficiently of the particles with significant pH sensitivity. Kinetics of drug release suggested self-sustained QUE release following the non-fickian trend. A pronounced hypoglycaemic effect and efficient maintenance of glucose homeostasis was evident in diabetic rat after peroral delivery of these quercetin nanoparticles in comparison to free oral quercetin. This suggests the fabrication of an efficient carrier of oral quercetin for diabetes treatment.

Alginate coated chitosan core-shell nanoparticles for efficient oral delivery of naringenin in diabetic animals-An in vitro and in vivo approach.

The chemical synthesis of this study targets for development of a bio-safe polymeric nano-vehicle for improvising the solubility of the flavanone naringenin in antidiabetic animal study. Nanoparticles were prepared from two cost-effective carbohydrate biopolymers - chitosan and alginate for successful encapsulation of naringenin. Dual crosslinked nanoparticles were synthesized by using Na2SO4 and CaCl2 as crosslinkers. The nanoparticles were characterized by DLS, FTIR, XRD and SEM. The prepared nano-formulations exhibited significant naringenin entrapment of >90% and pH-responsive slow and sustained release of the flavonoid. In-vivo studies revealed significant hypoglycemic effect after oral delivery of the nanoparticles to streptozotocin-induced diabetic rats. Histopathology and several blood parameters indicated that oral administrations of nanoparticles were free from toxicity. Other studies also suggested that polymeric formulations were quite effective for oral delivery of the flavonoid as a therapeutic agent in the treatment of dyslipidemia, hyperglycemia and haemoglobin iron-mediated oxidative stress in type 1 diabetic model.

Methylglyoxal-induced modification causes aggregation of myoglobin.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200µM) on the monomeric heme protein myoglobin (Mb) (100µM) in a time-dependent manner (7 to 18days incubation at 25°C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased ß-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

Copper induced immunotoxicity promote differential apoptotic pathways in spleen and thymus.

Inorganic copper, such as that in drinking water and copper supplements, largely bypasses the liver and enters the free copper pool of the blood directly and that promote immunosuppression. Nevertheless, the signaling pathways underlying copper-induced immune cell death remains largely unclear. According to our previous in vivo report, to evaluate the further details of the apoptotic mechanism, we have investigated how copper regulates apoptotic pathways in spleen and thymus. We have analyzed different protein expression by western blotting and immunohistochemistry and mRNA expression by RT-PCR and gel electrophoresis. We also have measured mitochondrial trans-membrane potential, ROS and CD4(+) and CD8(+) population by flow cytometry. Sub lethal doses of copper in spleen and thymus of in vivo Swiss albino mice promote different apoptotic pathways. In case of spleen, ROS generation and mitochondrial trans-membrane potential changes promotes intrinsic pathway of apoptosis that was p53 independent, ultimately leads to decrease in CD4(+) T cell population and increase in CD8(+) T cell population. However in case of thymus, ROS generation and mitochondrial trans-membrane potential changes lead to death receptor that regulate extrinsic and intrinsic pathways of apoptosis and the apoptotic mechanism which was p53 dependent. Due to copper treatment, thymic CD4(+) T cell population decreased and CD8(+) T cell population was increased or proliferated. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules like p27, p73, p62, poly (ADP-ribose) polymerase (PARP) that differentially changed due to copper treatment in spleen and thymus of Swiss albino mice. Present study firstly demonstrates how apoptotic pathways differentially regulate copper induced immunosuppression.