Masaud Shah

Middle East respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control

Middle East respiratory syndrome coronavirus (MERS-CoV) causes high fever, cough, acute respiratory tract infection and multiorgan dysfunction that may eventually lead to the death of the infected individuals. MERS-CoV is thought to be transmitted to humans through dromedary camels. The occurrence of the virus was first reported in the Middle East and it subsequently spread to several parts of the world. Since 2012, about 1368 infections, including ~487 deaths, have been reported worldwide. Notably, the recent human-to-human ‘superspreading’ of MERS-CoV in hospitals in South Korea has raised a major global health concern. The fatality rate in MERS-CoV infection is four times higher compared with that of the closely related severe acute respiratory syndrome coronavirus infection. Currently, no drug has been clinically approved to control MERS-CoV infection. In this study, we highlight the potential drug targets that can be used to develop anti-MERS-CoV therapeutics.

Insights into the species-specific TLR4 signaling mechanism in response to Rhodobacter sphaeroides lipid A detection

TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123–129) and loop EF (81–89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

In silico mechanistic analysis of IRF3 inactivation and high-risk HPV E6 species-dependent drug response

The high-risk human papillomavirus E6 (hrHPV E6) protein has been widely studied due to its implication in cervical cancer. In response to viral threat, activated kinases phosphorylate the IRF3 autoinhibitory domain, inducing type1 interferon production. HPV circumvents the antiviral response through the possible E6 interaction with IRF3 and abrogates p53’s apoptotic activity by recruiting E6-associated protein. However, the molecular mechanism of IRF3 inactivation by hrHPV E6 has not yet been delineated. Therefore, we explored this mechanism through in silico examination of protein-protein and protein-ligand docking, binding energy differences, and computational alanine mutagenesis. Our results suggested that the LxxLL motifs of IRF3 binds within the hydrophobic pocket of E6, precluding Ser-patch phosphorylation, necessary for IRF3 activation and interferon induction. This model was further supported by molecular dynamics simulation. Furthermore, protein-ligand docking and drug resistance modeling revealed that the polar patches in the pocket of E6, which are crucial for complex stability and ligand binding, are inconsistent among hrHPV species. Such variabilities pose a risk of treatment failure owing to point mutations that might render drugs ineffective, and allude to multi-drug therapy. Overall, this study reveals a novel perspective of innate immune suppression in HPV infections and suggests a plausible therapeutic intervention.

A structural insight into the negative effects of opioids in analgesia by modulating the TLR4 signaling: An in silico approach

Opioids are considered the gold standard therapy for pain. However, TLR-dependent negative effects in analgesia have highlighted the complexities in the pharmacodynamics of opioids. While successive studies have reported that morphine and Morphine-3-glucuronide (M3G) activate the TLR4 pathway, the structural details of this mechanism are lacking. Here, we have utilized various computational tools to reveal the structural dynamics of the opioid-bound TLR4/MD2 complex, and have proposed a potential TLR4 activation mechanism. Our results support previous findings, and include the novel insight that the stable binding of morphine and naloxone, but not M3G, in the MD2 cavity, is TLR4 dependent. Morphine interacts with MD2 near its Phe126 loop to induce the active conformation (MD2C); however, this binding is likely reversible, and the complex gains stability upon interaction with TLR4. M3G also induces the MD2C state, with both the Phe126 loop and the H1 loop being involved in MD2-M3G complex stability. Remarkably, naloxone, which requires TLR4 interaction for complex stability, switches the conformation of the gating loop to the inactive state (MD2°). Cumulatively, our findings suggest that ligand binding and receptor clustering occur successively in opioid-induced TLR4 signaling, and that MD2 plasticity and pocket hydrophobicity are crucial for the recognition and accommodation of ligands.

Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes.

The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming.

Structural and conformational insights into SOX2/OCT4-bound enhancer DNA: a computational perspective

The potential role of sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) are increasingly discussed in stem cell maintenance either in the context of iPSCs (induced pluripotent stem cells) generation or cancer stem cell growth. These proteins bind to the enhancer and drive the transcription of a multitude of other factors that facilitate stem cell propagation. Here, we elucidated the mechanism of changes in DNA shape and the precise role of the interaction with the proteins, which is necessary to manipulate this ternary complex. Besides bending the DNA, SOX2 drove the DNA into the A-form, whereas OCT4 preferentially shaped DNA into a B-like conformation. SOX2 binding expanded the minor groove with simultaneous shrinkage of the major grove. Greater fluctuation in the DNA and bound proteins was observed after disruption of the protein–protein interaction. Dynamic cross-correlation of DNA atoms was found to be variable, and entropy of DNA atoms from DNA-wild-type-SOX2/OCT4 (DNAWT) was the lowest among the various complexes. Moreover, essential dynamics-based conformational analysis revealed vivid conformational variation both in DNA alone and in protein bound complexes. Physical parameters such as the diffusion coefficient and dipole moment were also substantially different for DNA from the DNAWT complex. Taken together, our results establish a link between protein–protein and protein–DNA interactions, which will facilitate devising various strategies to modulate this complex in order to regulate the transcription of various proteins.

Advances in Antiviral Therapies Targeting Toll-like Receptors

ABSTRACT Introduction: Organisms have evolved a rapid and non-specific way to defend themselves via Toll-like receptors (TLRs), which recognize specific signatures present on invading microbes and viruses. Once detected, these receptors flood the cell with cytokines and IFNs that not only help to eradicate the invading viruses but also activate the adaptive immune response. Owing to difficulties in viral detection, a whole class of TLRs is dedicated to sensing viral nucleic acids, while other TLRs detect viral coat proteins and aid in establishing antiviral immunity. To protect humans better, TLRs and their downstream mediators can be used as potential drug targets, which can be either activated or inhibited, to counter viral infections. Areas covered: The current review focuses on TLR-targeting investigational drugs developed to treat viral diseases and virus-induced complications. Expert opinion: TLRs are a good choice for eradicating viral infections because they can fine-tune the immune response. However, TLRs should be exploited carefully, as there have been instances where their activation has led to unwanted responses in terms of both immune and viral activation. Therefore, more focus should be placed on novel drugs that can induce significant and long-term immunity, while concomitantly alleviating side effects.

Structural insights into the Middle East respiratory syndrome coronavirus 4a protein and its dsRNA binding mechanism

Middle East respiratory syndrome coronavirus (MERS-CoV) has evolved to navigate through the sophisticated network of a host’s immune system. The immune evasion mechanism including type 1 interferon and protein kinase R-mediated antiviral stress responses has been recently attributed to the involvement of MERS-CoV protein 4a (p4a) that masks the viral dsRNA. However, the structural mechanism of how p4a recognizes and establishes contacts with dsRNA is not well explained. In this study, we report a dynamic mechanism deployed by p4a to engage the viral dsRNA and make it unavailable to the host immune system. Multiple variants of p4a-dsRNA were created and investigated through extensive molecular dynamics procedures to highlight crucial interfacial residues that may be used as potential pharmacophores for future drug development. The structural analysis revealed that p4a exhibits a typical αβββα fold structure, as found in other dsRNA-binding proteins. The α1 helix and the β1-β2 loop play a crucial role in recognizing and establishing contacts with the minor grooves of dsRNA. Further, mutational and binding free energy analyses suggested that in addition to K63 and K67, two other residues, K27 and W45, might also be crucial for p4a-dsRNA stability.

Structural-dynamic insights into the H. pylori cytotoxin-associated gene A (CagA) and its abrogation to interact with the tumor suppressor protein ASPP2 using decoy peptides

Abstract Helicobacter pylori (H. pylori) is one of the most extensively studied Gram-negative bacteria due to its implication in gastric cancer. The oncogenicity of H. pylori is associated with cytotoxin-associated gene A (CagA), which is injected into epithelial cells lining the stomach. Both the C- and N-termini of CagA are involved in the interaction with several host proteins, thereby disrupting vital cellular functions, such as cell adhesion, cell cycle, intracellular signal transduction, and cytoskeletal structure. The N-terminus of CagA interacts with the tumor-suppressing protein, apoptosis-stimulating protein of p53 (ASPP2), subsequently disrupting the apoptotic function of tumor suppressor gene p53. Here, we present the in-depth molecular dynamic mechanism of the CagA–ASPP2 interaction and highlight hot-spot residues through in silico mutagenesis. Our findings are in agreement with previous studies and further suggest other residues that are crucial for the CagA–ASPP2 interaction. Furthermore, the ASPP2-binding pocket possesses potential druggability and could be engaged by decoy peptides, identified through a machine-learning system and suggested in this study. The binding affinities of these peptides with CagA were monitored through extensive computational procedures and reported herein. While CagA is crucial for the oncogenicity of H. pylori, our designed peptides possess the potential to inhibit CagA and restore the tumor suppressor function of ASPP2.

Adenylate kinase potentiates the capsular polysaccharide by modulating Cps2D in Streptococcus pneumoniae D39

Streptococcus pneumoniae is a polysaccharide-encapsulated bacterium. The capsule thickens during blood invasion compared with the thinner capsules observed in asymptomatic nasopharyngeal colonization. However, the underlying mechanism regulating differential CPS expression remains unclear. CPS synthesis requires energy that is supplied by ATP. Previously, we demonstrated a correlation between ATP levels and adenylate kinase in S. pneumoniae (SpAdK). A dose-dependent induction of SpAdK in serum was also reported. To meet medical needs, this study aimed to elucidate the role of SpAdK in the regulation of CPS production. CPS levels in S. pneumoniae type 2 (D39) increased proportionally with SpAdK levels, but they were not related to pneumococcal autolysis. Moreover, increased SpAdK levels resulted in increased total tyrosine kinase Cps2D levels and phosphorylated Cps2D, which is a regulator of CPS synthesis in the D39 strain. Our results also indicated that the SpAdK and Cps2D proteins interact in the presence of Mg-ATP. In addition, in silico analysis uncovered the mechanism behind this protein–protein interaction, suggesting that SpAdK binds with the Cps2D dimer. This established the importance of the ATP-binding domain of Cps2D. Taken together, the biophysical interaction between SpAdK and Cps2D plays an important role in enhancing Cps2D phosphorylation, which results in increased CPS synthesis.Pneumococcal disease: enzyme interaction promotes thickening of defensive bacterial capsuleA physical interaction between two key enzymes explains how the bacterium responsible for causing pneumococcal disease thickens its external capsule during infection of the bloodstream. A team led by Dong-Kwon Rhee from Sungkyunkwan University in Suwon, South Korea, studied strains of Streptococcus pneumoniae expressing varying levels of an enzyme that helps maintain the proper balance of cellular energy. They found that this enzyme stimulated the production of sugar chains that coat the outside of the bacterial capsule by binding and activating an intermediary enzyme involved in the synthesis of these sugar chains. Since the capsule is critical in warding off the human immune response, the findings suggest that drugs designed to disrupt the enzyme-mediated induction of capsule formation could help prevent or treat pneumococcal disease.