Protonation-State Dependent Modulation of Hen Egg-White Lysozyme Fibrillation under the Influence of a Short Synthetic Peptide.

Under the influence of various conditions, misfolding of soluble proteins occurs, leading to the formation of toxic insoluble amyloids. The formation and deposition of such amyloids within the body are associated with detrimental biological consequences such as the onset of several amyloid-related diseases. Previously, we established a strategy for the rational design of peptide inhibitors against amyloid formation based on the amyloidogenic-prone region of the protein. In the current study, we have designed and identified an Asp-containing rationally designed hexapeptide (SqP4) as an excellent inhibitor of hen egg-white lysozyme (HEWL) amyloid progression in vitro. First, SqP4 showed strong affinity toward the native monomeric HEWL leading to the stabilization of the native form and restriction in the unfolding process of monomeric HEWL. Second, SqP4 was found to arrest the amyloidogenic misfolded structure of HEWL in a nonfibrillar monomer-like stage. We also observed the differential effect of the protonation state of the charged amino acid (Asp) within the peptide inhibitor on the amyloid formation of HEWL and explored the reason behind the observations. The findings of this study can be implemented in future strategies for the development of potent therapeutics against other amyloid-related diseases.

Emerging trends and therapeutic applications of monoclonal antibodies.

Monoclonal antibodies (mAbs) are being used to prevent, detect, and treat a broad spectrum of malignancies and infectious and autoimmune diseases. Over the past few years, the market for mAbs has grown exponentially. They have become a significant part of many pharmaceutical product lines, and more than 250 therapeutic mAbs are undergoing clinical trials. Ever since the advent of hybridoma technology, antibody-based therapeutics were realized using murine antibodies which further progressed into humanized and fully human antibodies, reducing the risk of immunogenicity. Some of the benefits of using mAbs over conventional drugs include a drastic reduction in the chances of adverse reactions, interactions between drugs, and targeting specific proteins. While antibodies are very efficient, their higher production costs impede the process of commercialization. However, their cost factor has been improved by developing biosimilar antibodies, which are affordable versions of therapeutic antibodies. Along with biosimilars, innovations in antibody engineering have helped to design bio-better antibodies with improved efficacy than the conventional ones. These novel mAb-based therapeutics are set to revolutionize existing drug therapies targeting a wide spectrum of diseases, thereby meeting several unmet medical needs. In the future, mAbs generated by applying next-generation sequencing (NGS) are expected to become a powerful tool in clinical therapeutics. This article describes the methods of mAb production, pre-clinical and clinical development of mAbs, approved indications targeted by mAbs, and novel developments in the field of mAb research.

4-(Benzyloxy)phenol-induced p53 exhibits antimycobacterial response triggering phagosome-lysosome fusion through ROS-dependent intracellular Ca2+ pathway in THP-1 cells.

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 µM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.

Network pharmacology combined with molecular docking and in vitro verification reveals the therapeutic potential of Delphinium roylei munz constituents on breast carcinoma.

Delphinium roylei Munz is an indigenous medicinal plant to India where its activity against cancer has not been previously investigated, and its specific interactions of bioactive compounds with vulnerable breast cancer drug targets remain largely unknown. Therefore, in the current study, we aimed to evaluate the anti-breast cancer activity of different extracts of D. roylei against breast cancer and deciphering the molecular mechanism by Network Pharmacology combined with Molecular Docking and in vitro verification. The experimental plant was extracted with various organic solvents according to their polarity index. Phytocompounds were identified by High resolution-liquid chromatography-mass spectrometry (HR-LC/MS) technique, and SwissADME programme evaluated their physicochemical properties. Next, target(s) associated with the obtained bioactives or breast cancer-related targets were retrieved by public databases, and the Venn diagram selected the overlapping targets. The networks between overlapping targets and bioactive were visualized, constructed, and analyzed by STRING programme and Cytoscape software. Finally, we implemented a molecular docking test (MDT) using AutoDock Vina to explore key target(s) and compound(s). HR-LC/MS detected hundreds of phytocompounds, and few were accepted by Lipinski's rules after virtual screening and therefore classified as drug-like compounds (DLCs). A total of 464 potential target genes were attained for the nine quantitative phytocompounds and using Gene Cards, OMIM and DisGeNET platforms, 12063 disease targets linked to breast cancer were retrieved. With Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment, a total of 20 signalling pathways were manifested, and a hub signalling pathway (PI3K-Akt signalling pathway), a key target (Akt1), and a key compound (8-Hydroxycoumarin) were selected among the 20 signalling pathways via molecular docking studies. The molecular docking investigation revealed that among the nine phytoconstituents, 8-hydroxycoumarin showed the best binding energy (-9.2 kcal/mol) with the Akt1 breast cancer target. 8-hydroxycoumarin followed all the ADME property prediction using SwissADME, and 100 nanoseconds (ns) MD simulations of 8-hydroxycoumarin complexes with Akt1 were found to be stable. Furthermore, D. roylei extracts also showed significant antioxidant and anticancer activity through in vitro studies. Our findings indicated for the first time that D. roylei extracts could be used in the treatment of BC.

Soybean lectin-triggered IL-6 secretion induces autophagy to kill intracellular mycobacteria through P2RX7 dependent activation of the JAK2/STAT3/Mcl-1 pathway.

Cytokine therapy and cytokine-mediated autophagy have been used as prominent host-directed therapy (HDT) approaches to restrain M. tb growth in the host cell. In the present study, we have dissected the anti-tubercular activity of Soybean lectin (SBL) through cytokine-mediated autophagy induction in differentiated THP-1 (dTHP-1) cells. A significant increase in IL-6 expression was observed in both uninfected and mycobacteria infected dTHP-1 cells through the P2RX7 mediated pathway via PI3K/Akt/CREB-dependent signalling after SBL treatment. Inhibition of IL-6 level using IL-6 neutralizing antibody or associated signalling significantly enhanced the mycobacterial load in SBL-treated dTHP-1 cells. Further, autocrine signalling of IL-6 through its receptor-induced Mcl-1 expression activated autophagy via JAK2/STAT3 pathway, and inhibition of this pathway affected autophagy. Finally, blocking the IL-6-regulated autophagy through NSC 33994 (a JAK2 inhibitor) or S63845 (an Mcl-1 inhibitor) led to a notable increase in intracellular mycobacterial growth in SBL-treated cells. Taken together, these results indicate that SBL interacts with P2RX7 to regulate PI3K/Akt/CREB network to release IL-6 in dTHP-1 cells. The released IL-6, in turn, activates the JAK2/STAT3/Mcl-1 pathway upon interaction with IL-6Rα to modulate autophagy that ultimately controls mycobacterial growth in macrophages.

Ac-93253 inhibits intracellular growth of mycobacteria in human macrophages by inducing apoptosis in mitochondrial-dependent manner.

Recent studies suggest that apoptosis in macrophages plays a significant role in host defence against intracellular pathogens like viruses, fungi, protozoan, and bacteria, including Mycobacterium tuberculosis (M. tb). It is still unclear if micromolecules inducing apoptosis could be an attractive approach to combat the intracellular burden of M. tb. Hence, the present study has investigated the anti-mycobacterial effect of apoptosis mediated through phenotypic screening of micromolecules. Through MTT and trypan blue exclusion assay, 0.5 µM of Ac-93253 was found to be non-cytotoxic even after 72 h of treatment in phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. Significant regulation in the expression of various pro-apoptotic genes like Bcl-2, Bax, and Bad and the cleaved caspase 3 was observed upon treatment with a non-cytotoxic dose of Ac-93253. Ac-93253 treatment also leads to DNA fragmentation and increased phosphatidylserine accumulation in the plasma membrane's outer leaflet. Further, Ac-93253 also effectively reduced the growth of mycobacteria in infected macrophages, Z-VAD-FMK a broad-range apoptosis inhibitor significantly brought back the mycobacterial growth in Ac-93253 treated macrophages. These findings suggest apoptosis may be the probable effector response through which Ac-93253 manifests its anti-mycobacterial property.

Molecular Chaperones' Potential against Defective Proteostasis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neuronal degenerative condition identified via a build-up of mutant aberrantly folded proteins. The native folding of polypeptides is mediated by molecular chaperones, preventing their pathogenic aggregation. The mutant protein expression in ALS is linked with the entrapment and depletion of chaperone capacity. The lack of a thorough understanding of chaperones' involvement in ALS pathogenesis presents a significant challenge in its treatment. Here, we review how the accumulation of the ALS-linked mutant FUS, TDP-43, SOD1, and C9orf72 proteins damage cellular homeostasis mechanisms leading to neuronal loss. Further, we discuss how the HSP70 and DNAJ family co-chaperones can act as potential targets for reducing misfolded protein accumulation in ALS. Moreover, small HSPB1 and HSPB8 chaperones can facilitate neuroprotection and prevent stress-associated misfolded protein apoptosis. Designing therapeutic strategies by pharmacologically enhancing cellular chaperone capacity to reduce mutant protein proteotoxic effects on ALS pathomechanisms can be a considerable advancement. Chaperones, apart from directly interacting with misfolded proteins for protein quality control, can also filter their toxicity by initiating strong stress-response pathways, modulating transcriptional expression profiles, and promoting anti-apoptotic functions. Overall, these properties of chaperones make them an attractive target for gaining fundamental insights into misfolded protein disorders and designing more effective therapies against ALS.

Stonin 2 activates lysosomal-mTOR axis for cell survival in oral cancer.

Aberrant expression of various genes is associated with the progression of oral squamous cell carcinoma. Stonin 2, an endocytic protein, has a prominent role in clathrin-associated endocytosis. Its position in oral cancer is still unknown. Here, we report that STON2 expression increases with an increase in the grade of the oral cancer tissue. Further, STON2 overexpressed cells possess a higher rate of proliferation and migraton in oral cancer cells. STON2 helps maintain lysosomal functions by preserving the lysosomal membrane integrity. It activates the Akt-mTOR axis and retains the mTOR on the membrane of the lysosomes. Further, we have identified an inhibitor of STON2, i.e., Trifluoperazine dihydrochloride (TFP), which targets the lysosomal axis by disrupting the Akt-mTOR pathway and causes lysosomal membrane permeabilization. Intererstingly, TFP shows a decrease in cell vaibility on the oral cancer cells and it was observed that cell viability is restored in TFP-treated STON2 overexpressed cells. Moreover, the lysosomal activity and the Akt-mTOR expression are restored in STON2 overexpressed cells co-treated with TFP, establishing TFP targets STON2 to showcase its anti-cancer effects in oral cancer. In conclusion, STON2 might serve as a potential biomarker in oral cancer, and its inhibition could functions as a novel anti-cancer mechanims against oral cancer.

Development and Challenges of Diclofenac-Based Novel Therapeutics: Targeting Cancer and Complex Diseases.

Diclofenac is a highly prescribed non-steroidal anti-inflammatory drug (NSAID) that relieves inflammation, pain, fever, and aches, used at different doses depending on clinical conditions. This drug inhibits cyclooxygenase-1 and cyclooxygenase-2 enzymes, which are responsible for the generation of prostaglandin synthesis. To improve current diclofenac-based therapies, we require new molecular systematic therapeutic approaches to reduce complex multifactorial effects. However, the critical challenge that appears with diclofenac and other drugs of the same class is their side effects, such as signs of stomach injuries, kidney problems, cardiovascular issues, hepatic issues, and diarrhea. In this article, we discuss why defining diclofenac-based mechanisms, pharmacological features, and its medicinal properties are needed to direct future drug development against neurodegeneration and imperfect ageing and to improve cancer therapy. In addition, we describe various advance molecular mechanisms and fundamental aspects linked with diclofenac which can strengthen and enable the better designing of new derivatives of diclofenac to overcome critical challenges and improve their applications.

Autophagy based cellular physiological strategies target oncogenic progression.

Evidence accumulated from past findings indicates that defective proteostasis may contribute to risk factors for cancer generation. Irregular assembly of abnormal proteins catalyzes the disturbance of cellular proteostasis and induces the ability of abnormal cellular proliferation. The autophagy mechanism plays a key role in the regular clearance of abnormal/poor lipids, proteins, and various cellular organelles. The results of functional and effective autophagy deliver normal cellular homeostasis, which establishes supportive metabolism and avoids unexpected tumorigenesis events. Still, the precise molecular mechanism of autophagy in tumor suppression has not been clear. How autophagy triggers selective or nonselective bulk degradation to dissipate tumor promotion under stress conditions is not clear. Under proteotoxic insults to knockdown the drive of tumorigenesis, it is critical for us to figure out the detailed molecular functions of autophagy in human cancers. The current article summarizes autophagy-based theragnostic strategies targeting various phases of tumorigenesis and suggests the preventive roles of autophagy against tumor progression. A better understanding of various molecular partners of autophagic flux will improve and innovate therapeutic approaches based on autophagic-susceptible effects against cellular oncogenic transformation.

Inflammasomes in cancer: Effect of epigenetic and autophagic modulations.

Tumour-promoting inflammation is a critical hallmark in cancer development, and inflammasomes are well-known regulators of inflammatory processes within the tumour microenvironment. Different inflammasome components along with the adaptor, apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC), and the effector, caspase-1, have a significant influence on tumorigenesis but in a tissue-specific and stage-dependent manner. The downstream products of inflammasome activation, that is the proinflammatory cytokines such as IL-1ß and IL-18, regulate tissue homeostasis and induce antitumour immune responses, but in contrast, they can also favour cancer growth and proliferation by directing various oncogenic signalling pathways in cancer cells. Moreover, different epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNAs, control inflammasomes and their components by regulating gene expression during cancer progression. Furthermore, autophagy, a master controller of cellular homeostasis, targets inflammasome-induced carcinogenesis by maintaining cellular homeostasis and removing potential cancer risk factors that promote inflammasome activation in support of tumorigenesis. Here, in this review, we summarize the effect of inflammasome activation in cancers and discuss the role of epigenetic and autophagic regulatory mechanisms in controlling inflammasomes. A proper understanding of the interactions among these key processes will be useful for developing novel therapeutic regimens for targeting inflammasomes in cancer.

Complex Inclusion Bodies and Defective Proteome Hubs in Neurodegenerative Disease: New Clues, New Challenges.

A healthy physiological environment of cells represents the dynamic homeostasis of crowded molecules. A subset of cellular proteome forms protein quality control (PQC) machinery to maintain an uninterrupted synthesis of new polypeptides and targeted elimination of old or defective proteins. The process of PQC may get overwhelmed under specific genetic mutations, environmental stress conditions, and aging-associated perturbances. Many of these conditions may lead to the generation of various types of aberrant protein species that may or may not accumulate as large cellular inclusions. These proteinaceous formations, referred to as inclusion bodies (IBs), could be membrane-bound or membrane-less, cytoplasmic, or nuclear. Most importantly, they could either be toxic or protective. Under acute stress conditions, the formation of aggregates may cause proteostasis failure, leading to large-scale changes in the cellular proteome compositions. However, the large insoluble IBs may act as reservoirs for many soluble proteins with high aggregation propensities, which can overwhelm the cellular chaperoning capacity and protein degradation machinery. The kinetic equilibrium between folding and unfolding, misfolding, and refolding; aggregation and degradation is perturbed in one or many neurodegenerative disorders (NDDs) associated with dementia, cognitive impairments, movement, and behavioural losses. However, a detailed interplay of IBs into the manifestation of the NDDs is unknown, and a very primitive knowledge of structural compositions of amyloid inclusions is present. The present article presents a brief evolutionary background of IBs; their functional relevance for prokaryotes, plants, and animals; and associated involvement in neuronal proteostasis.

P2X7 receptor in multifaceted cellular signalling and its relevance as a potential therapeutic target in different diseases.

P2X7 receptor, a purinergic receptor family member, is abundantly expressed on many cells, including immune, muscle, bone, neuron, and glia. It acts as an ATP-activated cation channel that permits the influx of Ca2+, Na+ and efflux of K+ ions. The P2X7 receptor plays crucial roles in many physiological processes including cytokine and chemokine secretion, NLRP3 inflammasome activation, cellular growth and differentiation, locomotion, wound healing, transcription factors activation, cell death and T-lymphocyte survival. Past studies have demonstrated the up-regulation and direct association of this receptor in many pathophysiological conditions such as cancer, diabetics, arthritis, tuberculosis (TB) and inflammatory diseases. Hence, targeting this receptor is considered a worthwhile approach to lessen the afflictions associated with the disorders mentioned above by understanding the receptor architecture and downstream signalling processes. Here, in the present review, we have dissected the structural and functional aspects of the P2X7 receptor, emphasizing its role in various diseased conditions. This information will provide in-depth knowledge about the receptor and help to develop apt curative methodologies for the betterment of humanity in the coming years.

Ibuprofen-based advanced therapeutics: breaking the inflammatory link in cancer, neurodegeneration, and diseases.

Ibuprofen is a classical nonsteroidal anti-inflammatory drug (NSAID) highly prescribed to reduce acute pain and inflammation under an array of conditions, including rheumatoid arthritis, osteoarthritis, dysmenorrhea, and gout. Ibuprofen acts as a potential inhibitor for cyclooxygenase enzymes (COX-1 and COX-2). In the past few decades, research on this small molecule has led to identifying other possible therapeutic benefits. Anti-tumorigenic and neuroprotective functions of Ibuprofen are majorly recognized in recent literature and need further consideration. Additionally, several other roles of this anti-inflammatory molecule have been discovered and subjected to experimental assessment in various diseases. However, the major challenge faced by Ibuprofen and other drugs of similar classes is their side effects, and tendency to cause gastrointestinal injury, generate cardiovascular risks, modulate hepatic and acute kidney diseases. Future research should also be conducted to deduce new methods and approaches of suppressing the unwanted toxic changes mediated by these drugs and develop new therapeutic avenues so that these small molecules continue to serve the purposes. This article primarily aims to develop a comprehensive and better understanding of Ibuprofen, its pharmacological features, therapeutic benefits, and possible but less understood medicinal properties apart from major challenges in its future application.KEY POINTSIbuprofen, an NSAID, is a classical anti-inflammatory therapeutic agent.Pro-apoptotic roles of NSAIDs have been explored in detail in the past, holding the key in anti-cancer therapies.Excessive and continuous use of NSAIDs may have several side effects and multiple organ damage.Hyperactivated Inflammation initiates multifold detrimental changes in multiple pathological conditions.Targeting inflammatory pathways hold the key to several therapeutic strategies against many diseases, including cancer, microbial infections, multiple sclerosis, and many other brain diseases.

Soybean lectin induces autophagy through P2RX7 dependent activation of NF-κB-ROS pathway to kill intracellular mycobacteria.

BACKGROUND: Host-directed therapy is considered a novel anti-tuberculosis strategy in tackling the tuberculosis burden through autophagy induction by various inducers to curtail the growth of intracellular Mycobacterium tuberculosis. METHODS: In this study, we investigated the anti-tubercular role of soybean lectin, a lectin isolated from Glycine max (Soybean). Effect of SBL on intracellular mycobacterial viability through autophagy and the mechanism involved in differentiated THP-1 cells was studied using different experimental approaches. RESULTS: We initially performed a time kinetic experiment with the non-cytotoxic dose of SBL (20 µg/ml) and observed autophagy induction after 24 h of treatment. Abrogation of autophagy in the presence of 3-MA and an increase in LC3 puncta formation upon Baf-A1 addition elucidated the specific effect on autophagy and autophagic flux. SBL treatment also led to autophagy induction in mycobacteria infected macrophages that restricted the intracellular mycobacterial growth, thus emphasizing the host defensive role of SBL induced autophagy. Mechanistic studies revealed an increase in P2RX7 expression, NF-κB activation and reactive oxygen species generation upon SBL treatment. Inhibition of P2RX7 expression suppressed NF-κB dependent ROS level in SBL treated cells. Moreover, SBL induced autophagy was abrogated in the presence of either different inhibitors or P2RX7 siRNA, leading to the reduced killing of intracellular mycobacteria. CONCLUSION: Taken together, these results conclude that SBL induced autophagy exerts an anti-mycobacterial effect in P2RX7-NF-κB dependent manner through the generation of ROS. GENERAL SIGNIFICANCE: This study has provided a novel anti-mycobacterial role of SBL, which may play an important role in devising new therapeutic interventions.

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Mycobacteria Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Mycobacterium tuberculosis Pathogenesis.

Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-ß, IL-1α, and IL-1ß levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013171.

Structure-function and application of plant lectins in disease biology and immunity.

Lectins are proteins with a high degree of stereospecificity to recognize various sugar structures and form reversible linkages upon interaction with glyco-conjugate complexes. These are abundantly found in plants, animals and many other species and are known to agglutinate various blood groups of erythrocytes. Further, due to the unique carbohydrate recognition property, lectins have been extensively used in many biological functions that make use of protein-carbohydrate recognition like detection, isolation and characterization of glycoconjugates, histochemistry of cells and tissues, tumor cell recognition and many more. In this review, we have summarized the immunomodulatory effects of plant lectins and their effects against diseases, including antimicrobial action. We found that many plant lectins mediate its microbicidal activity by triggering host immune responses that result in the release of several cytokines followed by activation of effector mechanism. Moreover, certain lectins also enhance the phagocytic activity of macrophages during microbial infections. Lectins along with heat killed microbes can act as vaccine to provide long term protection from deadly microbes. Hence, lectin based therapy can be used as a better substitute to fight microbial diseases efficiently in future.

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

ESAT-6 modulates Calcimycin-induced autophagy through microRNA-30a in mycobacteria infected macrophages.

OBJECTIVE: Mycobacterium tuberculosis (M. tb) has a sumptuous repertoire of effector molecules to counter host defenses. Some of these antigens inhibit autophagy but the exact mechanism of this inhibition is poorly understood. METHODS: Purified protein derivative (PPD) was fractionated using 10 (PPD 10, antigenic molecular weight > 10 kDa) and 3 (PPD 3, mol. weight > 3 kDa) kDa cutters. Effect of these fractions on Calcimycin-induced autophagy and intracellular mycobacterial viability was then studied using different experimental approaches. RESULT: We found significant downregulation of autophagy by PPD 3 pre-treatment in Calcimycin-treated dTHP-1 cells compared to PPD 10. This reduction in autophagy also corroborated with the enhanced survival of mycobacteria in macrophages. We demonstrate that recombinant early secreted antigenic target 6 (rESAT-6) is responsible to inhibit Calcimycin-induced autophagy and enhance intracellular survival of mycobacteria. We also show that pre-treatment with rESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-a-vis downregulates miR-30a-5p expression in Calcimycin-treated dTHP-1 cells. Transfection studies with either miR-30a-3p inhibitor or miR-30a-5p mimic clearly elucidated the opposing roles of miR-30a-3p and miR-30a-5p in rESAT-6 mediated mycobacterial survival through autophagy inhibition. CONCLUSION: Taken together, our result evidently highlights that rESAT-6 enhances intracellular survival of mycobacteria by modulating miR-30a-3p and miR-30a-5p expression.

Paederia foetida induces anticancer activity by modulating chromatin modification enzymes and altering pro-inflammatory cytokine gene expression in human prostate cancer cells.

Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.