Role of MicroRNA-21 in Prostate Cancer Progression and Metastasis: Molecular Mechanisms to Therapeutic Targets.

The role of noncoding RNA has made remarkable progress in understanding progression, metastasis, and metastatic castration-resistant prostate cancer (mCRPC). A better understanding of the miRNAs has enhanced our knowledge of their targeting mainly at the therapy level in solid tumors, such as prostate cancer (PCa). microRNAs (miRNAs) belong to a class of endogenous RNA that deficit encoded proteins. Therefore, the role of miRNAs has been well-coined in the progression and development of PCa. miR-21 has a dual nature in its work both as a tumor suppressor and oncogenic role, but most of the recent studies showed that miR-21 is a tumor promoter and also is involved in castration-resistant prostate cancer (CRPC). Upregulation of miR-21 suppresses programmed cell death and inducing metastasis and castration resistant in PCa. miR-21 is involved in the different stages, such as proliferation, angiogenesis, migration, and invasion, and plays an important role in the progression, metastasis, and advanced stages of PCa. Recently, various studies directly linked the role of high levels of miR-21 with a poor therapeutic response in the patient of PCa. In the present review, we have explained the molecular mechanisms/pathways of miR-21 in PCa progression, metastasis, and castration resistant and summarized the role of miR-21 in diagnosis and therapeutic levels in PCa. In addition, we have spotlighted the recent therapeutic strategies for targeting different stages of PCa.

Structure-based virtual screening against multiple Plasmodium falciparum kinases reveals antimalarial compounds.

The continuous emergence of resistance against most frontline antimalarial drugs has led to countless deaths in malaria-endemic countries, counting 619,000 deaths in 2021, with mutation in drug targets being the sole cause. As mutation is correlated frequently with fitness cost, the likelihood of mutation emergence in multiple targets at a time is extremely low. Hence, multitargeting compounds may seem promising to address drug resistance issues with additional benefits like increased efficacy, improved safety profile, and the requirement of fewer pills compared to traditional single and combinational drugs. In this study, we attempted to use the High Throughput Virtual Screening approach to predict multitarget inhibitors against six chemically validated Plasmodium falciparum (Pf) kinases (PfPKG, PfMAP2, PfCDPK4, PfTMK, PfPK5, PfPI4K), resulting in 21 multitargeting hits. The molecular dynamic simulation of the top six complexes (Myricetin-MAP2, Quercetin-CDPK4, Myricetin-TMK, Quercetin-PKG, Salidroside-PK5, and Salidroside-PI4K) showed stable interactions. Moreover, hierarchical clustering reveals the structural divergence of the compounds from the existing antimalarials, indicating less chance of cross-resistance. Additionally, the top three hits were validated through parasite growth inhibition assays, with quercetin and myricetin exhibiting an IC50 value of 1.84 and 3.93 µM, respectively.

Widespread amyloid aggregates formation by Zika virus proteins and peptides.

Viral pathogenesis typically involves numerous molecular mechanisms. Protein aggregation is a relatively unknown characteristic of viruses, despite the fact that viral proteins have been shown to form terminally misfolded forms. Zika virus (ZIKV) is a neurotropic one with the potential to cause neurodegeneration. Its protein amyloid aggregation may link the neurodegenerative component to the pathogenicity associated with the viral infection. Therefore, we investigated protein aggregation in the ZIKV proteome as a putative pathogenic route and one of the alternate pathways. We discovered that it contains numerous anticipated aggregation-prone regions in this investigation. To validate our prediction, we used a combination of supporting experimental techniques routinely used for morphological characterization and study of amyloid aggregates. Several ZIKV proteins and peptides, including the full-length envelope protein, its domain III (EDIII) and fusion peptide, Pr N-terminal peptide, NS1 ß-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein, were shown to be highly aggregating in our study. Because our findings show that viral proteins can form amyloids in vitro, we need to do a thorough functional study of these anticipated APRs to understand better the role of amyloids in the pathophysiology of ZIKV infection.

The multi-protein targeting potential of bioactive syringin in inflammatory diseases: using molecular modelling and in-silico analysis of regulatory elements.

Inflammation plays a crucial role in the onset or progression of a variety of acute and chronic diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) are the only available FDA-approved therapy. The therapeutic outcome of NSAIDs is still finite due to off-target effects and extreme side effects on other vital organs. Bioactive syringin has been manifested to hold anti-osteoporosis, cardiac hypertrophy, alter autophagy, anti-cancer, neuro-preventive effects, etc. However, its multi-protein targeting potential in inflammation mostly remains unexplored. In the present work, we have checked the multi-protein targeting potential of bioactive glycoside syringin in inflammatory diseases. Based on the binding score of protein-ligand complexes, glycoside syringin scored greater than -7 kcal/mol against 12 inflammatory proteins. Our molecular dynamic simulation study (200 ns) confirmed that bioactive syringin remained inside the binding cavity of inflammatory proteins (JAK1, TYK2, and COX1) in a stable conformation. Further, our co-expression analysis suggests that these genes play an essential role in multiple pathways and are regulated by multiple miRNAs. Our study demonstrates that bioactive glycoside syringin might be a multi-protein targeting potential against inflammatory diseases and could be further investigated utilizing different preclinical approaches.Communicated by Ramaswamy H. Sarma.

Coronaviruses spike glycoprotein endodomains: The sequence and structure-based comprehensive study.

Any protein's flexibility or region makes it available to interact with many biomolecules in the cell. Specifically, such interactions in viruses help them to perform more functions despite having a smaller genome. Therefore, these flexible regions can be exciting and essential targets to be explored for their role in pathogenicity and therapeutic developments as they achieve essential interactions. In the continuation with our previous study on disordered analysis of SARS-CoV-2 spike protein's cytoplasmic tail (CTR), or endodomain, here we have explored the endodomain's disordered potential of six other coronaviruses using multiple bioinformatics approaches and molecular dynamics simulations. Based on the comprehensive analysis of its sequence and structural composition, we report the varying disorder propensity in endodomains of spike proteins of coronaviruses. The observations of this study may help to understand the importance of spike glycoprotein endodomain and creating therapeutic interventions against them.

Potential of ADAM 17 Signal Peptide To Form Amyloid Aggregates in Vitro.

ADAM 17, a disintegrin and metalloproteinase 17 belonging to the adamalysin protein family, is a Zn2+-dependent type-I transmembrane α-secretase protein. As a major sheddase, ADAM 17 acts as an indispensable regulator of chief cellular events and controls diverse cytokines, adhesion molecules, and growth factors. The signal peptide (residues 1-17) of ADAM 17 targets the protein to the secretory pathway and gets cleaved off afterward. No other function is documented for the ADAM 17 signal peptide (ADAM 17-SP) inside the cells. Here, we have taken a reductionist approach to understand the biophysical properties of ADAM 17-SP. Aiming to understand the possibility of aggregation, we found several aggregation-prone segments in the signal peptide. We performed in vitro experiments to show that the signal peptide forms amyloid-like aggregates in buffered conditions. We also studied its aggregation in the presence of sodium tripolyphosphate and heparin to correlate with the cellular conditions, as these biomolecules are naturally present inside cells. Further, we performed seeding experiments to observe the possibility of ADAM 17-SP aggregate interaction with the Aß42 peptide. The results suggest that its seeds escalate the aggregation kinetics of the Aß42 peptide and form heteromeric aggregates with it. We believe this finding could further intensify the aggregation studies on other signal peptides and shed light on the potential role of these segments other than signaling.

Structure-based virtual screening approach reveals natural multi-target compounds for the development of antimalarial drugs to combat drug resistance.

Compared to the previous year, there has been an increase of nearly 2 million malaria cases in 2021. The emergence of drug-resistant strains of Plasmodium falciparum, the most deadly malaria parasite, has led to a decline in the effectiveness of existing antimalarial drugs. To address this problem, the present study aimed to identify natural compounds with the potential to inhibit multiple validated antimalarial drug targets. The natural compounds from the Natural Product Activity and Species Source (NPASS) database were screened against ten validated drug targets of Plasmodium falciparum using a structure-based molecular docking method. Twenty compounds, with targets ranging from three to five, were determined as the top hits. The molecular dynamics simulations of the top six complexes (NPC246162 in complex with PfAdSS, PfGDH, and PfNMT; NPC271270 in complex with PfCK, PfGDH, and PfdUTPase) confirmed their stable binding affinity in the dynamic environment. The Tanimoto coefficient and distance matrix score analysis show the structural divergence of all the hit compounds from known antimalarials, indicating minimum chances of cross-resistance. Thus, we propose further investigating these compounds in biochemical and parasite inhibition studies to reveal the real therapeutic potential. If found successful, these compounds may be a new avenue for future drug discovery efforts to combat existing antimalarial drug resistance.Communicated by Ramaswamy H. Sarma.

Investigating the aggregation perspective of Dengue virus proteome.

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the ß-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities.

Immunomodulatory potential of bioactive glycoside syringin: a network pharmacology and molecular modeling approach.

Many diseases, such as rheumatoid arthritis, neurodegenerative disease, lupus, autoimmune disease, and cancer, are described by chronic inflammation following tissue damage. Anti-inflammatory drugs like non-steroidal anti-inflammatory drugs and other steroids cause many side effects and generally need careful consideration and monitoring during usage. In recent years, a significant interest in plant-derived approaches has been warranted. The bioactive glycoside syringin might be one of the effective immunomodulatory agents. However, its immunomodulatory potential needs to be better known. In this study, we evaluated the immunomodulatory potential of syringin using network pharmacology, molecular docking, and molecular dynamics simulation-based approaches. First, we applied the GeneCards and OMIM databases to acquire the immunomodulatory agents. Then, the STRING database was utilized to get the hub genes. Interaction analysis and molecular docking described strong binding of the active site of immunomodulatory proteins with the bioactive syringin. Molecular dynamics simulations (200 ns) showed a very stable interaction of syringin with the immunomodulatory protein. Further, the optimized structure and molecular electrostatic potential of the syringin were calculated by a density-functional theory utilizing basis levels of B3LYP/6-31. Syringin investigated in this study holds the required drug-likeness characteristics and follows Lipinski's rule of five. However, quantum-chemical estimations show the syringin has potent reactivity, demonstrating a lower energy gap. Furthermore, the gap between ELUMO and EHOMO was low, suggesting the excellent affinity of syringin towards the immunomodulatory proteins. The present study shows that syringin might be an effective immunomodulatory agent and can be further explored using different experimental methods.Communicated by Ramaswamy H. Sarma.

Investigating the folding dynamics of NS2B protein of Zika virus.

NS2B protein of the Zika virus acts as a co-factor for NS3 protease and also involves in remodeling NS3 protease structure. Therefore, we investigated the overall dynamics of NS2B protein. We find surprising similarities between selected flavivirus NS2B model structures predicted from Alphafold2. Further, the simulated ZIKV NS2B protein structure shows a disordered cytosolic domain (residues 45-95) as a part of a full-length protein. Since only the cytosolic domain of NS2B is sufficient for the protease activity, we also investigated the conformational dynamics of only ZIKV NS2B cytosolic domain (residues 49-95) in the presence of TFE, SDS, Ficoll, and PEG using simulation and spectroscopy. The presence of TFE induces α-helix in NS2B cytosolic domain (residues 49-95). On the other hand, the presence of SDS, ficoll, and PEG does not induce secondary structural change. This dynamics study could have implications for some unknown folds of the NS2B protein.

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes.

The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge of the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions (APRs) in these proteomes and revealed a strong aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV and SARS-CoV-2 proteins and protein regions, including the signal sequence peptide and fusion peptides 1 and 2 of the spike protein, a peptide from the NSP6 protein, and the ORF10 and NSP11 proteins. Our results show that these peptides and proteins can form amyloid aggregates. We used circular dichroism spectroscopy to reveal the presence of ß-sheet rich cores in aggregates and X-ray diffraction and Raman spectroscopy to confirm the formation of amyloid structures. Furthermore, we demonstrated that SARS-CoV-2 NSP11 aggregates are toxic to mammalian cell cultures. These results motivate further studies about the possible role of aggregation of SARS proteins in protein misfolding diseases and other human conditions.

Platelet-derived microvesicles activate human platelets via intracellular calcium mediated reactive oxygen species release.

Platelet-derived microvesicles (PMVs) are the most abundant microvesicles in circulation, originating from blood platelets via membrane blebbing. PMVs act as biological cargo carrying key molecules from platelets, including immunomodulatory molecules, growth factors, clotting molecules, and miRNAs that can regulate recipient cellular functions. Formation and release of PMVs play an essential role in the pathophysiology of vascular diseases such as hemostasis, inflammation, and thrombosis. Platelet activation is considered the critical event in thrombosis, and a growing number of evidence suggests that oxidative stress-mediated signaling plays a significant role in platelet activation. Ca2+ is a notable player in the generation of ROS in platelets. Reports have established that microvesicles exhibit dual nature in redox mechanisms as they possess both pro-oxidant and antioxidant machinery. However, the impact of PMVs and their ROS machinery on platelets is still a limited explored area. Here, we have demonstrated that PMVs mediate platelet activation via intracellular ROS generation. PMVs interacted with platelets and induced calcium-mediated intracellular ROS production via NADPH oxidase (NOX), leading to platelet activation. Our findings will open up new insights into the tangible relationship of PMVs with platelets and will further contribute to the therapeutic aspects of PMVs in vascular injury and tissue remodeling.

An insight into SARS-CoV-2 membrane protein interaction with spike, envelope, and nucleocapsid proteins.

Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, we tried to understand the interaction of membrane protein's interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies were performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid proteins. Prime MM-GBSA showed high binding energy calculations for the simulated structures than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.Communicated by Ramaswamy H. Sarma.

Investigating the conformational dynamics of Zika virus NS4B protein.

Zika virus (ZIKV) NS4B protein is a membranotropic multifunctional protein. Despite its versatile functioning, its topology and dynamics are not entirely understood. There is no X-ray or cryo-EM structure available for any flaviviral NS4B full-length protein. In this study, we have investigated the structural dynamics of full-length ZIKV NS4B protein through 3D structure models using molecular dynamics simulations and experimental techniques. Also, we employed a reductionist approach to understand the dynamics of NS4B protein where we studied its N-terminal (residues 1-38), C-terminal (residues 194-251), and cytosolic (residues 131-169) regions in isolation in addition to the full-length protein. Further, using a series of circular dichroism spectroscopic experiments, we validate the cytosolic region as an intrinsically disordered protein region. The microsecond-long all atoms molecular dynamics and replica-exchange simulations complement the experimental observations. Furthermore, we have also studied the NS4B proteins C-terminal regions of four other flaviviruses viz. DENV2, JEV, WNV, and YFV through microsecond simulations to characterize their behaviour in presence and absence of lipid membranes. There are significant differences observed in the conformations of other flavivirus NS4B C-terminal regions in comparison to ZIKV NS4B. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions.

p53 TAD2 Domain (38-61) Forms Amyloid-like Aggregates in Isolation.

A strong association between protein aggregation and human diseases (such as Alzheimer's, Parkinson's, and Huntington's disease) is well demonstrated. Misfolding and aggregation of p53, a central transcriptional mediator, has been revealed by various experimental evidence in different types of cancers. Aggregation studies focusing on different p53 domains, mostly, the central core domain and its mutants under the influence of various environmental conditions, and the p53 transactivation domain (TAD) (1-63) have been reported. However, the specific subdomains responsible for p53 aggregation are not known. p53 TADs interact with diverse cellular factors to modulate the function of p53 and elicit appropriate cellular responses under different stress conditions. In this study, the aggregation of the p53 TAD2 domain (38-61) has been studied in isolation. The aggregates were generated in vitro under acidic pH conditions after in silico scoring for amyloidogenic tendency and characterized using dye-based assays (ThT and bis-ANS fluorescence), CD spectroscopy, and microscopy (scanning electron microscoy, transmission electron microscopy, and atomic force microscopy). It was observed that p53 TAD2 forms characteristic ß-sheet-rich amyloid-like fibrils. Via a reductionist approach, this study highlights the nature of p53 TAD2 domain (38-61) aggregation.

Role of structural disorder in the multi-functionality of flavivirus proteins.

INTRODUCTION: The life cycle of a virus involves interacting with the host cell, entry, hijacking host machinery for viral replication, evading the host's immune system, and releasing mature virions. However, viruses, being small in size, can only harbor a genome large enough to code for the minimal number of proteins required for the replication and maturation of the virions. As a result, many viral proteins are multifunctional machines that do not directly obey the classic structure-function paradigm. Often, such multifunctionality is rooted in intrinsic disorder that allows viral proteins to interact with various cellular factors and remain functional in the hostile environment of different cellular compartments. AREAS COVERED: This report covers the classification of flaviviruses, their proteome organization, and the prevalence of intrinsic disorder in the proteomes of different flaviviruses. Further, we have summarized the speculations made about the apparent roles of intrinsic disorder in the observed multifunctionality of flaviviral proteins. EXPERT OPINION: Small sizes of viral genomes impose multifunctionality on their proteins, which is dependent on the excessive usage of intrinsic disorder. In fact, intrinsic disorder serves as a universal functional tool, weapon, and armor of viruses and clearly plays an important role in their functionality and evolution.

Structural dynamics of Zika virus NS1 via a reductionist approach reveal the disordered nature of its ß-roll domain in isolation.

Flavivirus Non-structural 1 (NS1) protein performs multiple functions and it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. It is important to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Therefore, we have employed extensively long molecular dynamic (MD) simulations in understanding the dynamics of the three structural domains (i.e., ß-roll, wing, and ß-ladder) in isolation, as a reductionist approach. We also found that the ß-ladder domain is highly flexible, while the ß-roll domain is disordered during long simulations. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 ß-roll in isolation and lipid mimetic environments. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1.

Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal.

One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of the NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed different spectroscopic technique-based binding assays of an anticancer drug mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular dynamics simulations of the docked complex with two different force fields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting the NSP1 protein of SARS-CoV-2.

Functional inhibition of c-Myc using novel inhibitors identified through "hot spot" targeting.

Protein-protein interactions drive various biological processes in healthy as well as disease states. The transcription factor c-Myc plays a crucial role in maintaining cellular homeostasis, and its deregulated expression is linked to various human cancers; therefore, it can be considered a viable target for cancer therapeutics. However, the structural heterogeneity of c-Myc due to its disordered nature poses a major challenge to drug discovery. In the present study, we used an in silico alanine scanning mutagenesis approach to identify "hot spot" residues within the c-Myc/Myc-associated factor X interface, which is highly disordered and has not yet been systematically analyzed for potential small molecule binding sites. We then used the information gained from this analysis to screen potential inhibitors using a conformation ensemble approach. The fluorescence-based biophysical experiments showed that the identified hit molecules displayed noncovalent interactions with these hot spot residues, and further cell-based experiments showed substantial in vitro potency against diverse c-Myc-expressing cancer/stem cells by deregulating c-Myc activity. These biophysical and computational studies demonstrated stable binding of the hit compounds with the disordered c-Myc protein. Collectively, our data indicated effective drug targeting of the disordered c-Myc protein via the determination of hot spot residues in the c-Myc/Myc-associated factor X heterodimer.