Covalent ligands of nuclear receptors.

Nuclear receptors (NRs) are ligand-induced transcriptional factors implicated in several physiological pathways. Naïve ligands bind to their cognate receptors and modulate gene expression as agonists or antagonists. It has been observed that some ligands bind via covalent bonding with the NR Ligand Binding Domain (LBD) residues. While many such instances have been known since the 1980s, a consolidated account of these ligands and their interactions with NR-LBD is yet to be documented. To negate this, we have culled out the human NR-LBDs that form a covalent attachment with ligands. According to the study, 16 of the 48 human NRs have been targeted by covalent ligands. It was found that conserved cysteines prone to covalent attachment are predominantly located in NR-LBD helices 3 and 11. These conserved cysteines are also observed in many of the remaining NRs, which can be probed for their reactivity. Thus, the structural insights into NR-LBD interactions with covalent ligands presented here would aid drug discovery efforts targeting NRs.

Structural insights into Plasmodium PPIases.

Malaria is one of the most prevalent infectious diseases posing a serious challenge over the years, mainly owing to the emergence of drug-resistant strains, sparking a need to explore and identify novel protein targets. It is a well-known practice to adopt a chemo-genomics approach towards identifying targets for known drugs, which can unravel a novel mechanism of action to aid in better drug targeting proficiency. Immunosuppressive drugs cyclosporin A, FK506 and rapamycin, were demonstrated to inhibit the growth of the malarial parasite, Plasmodium falciparum. Peptidyl prolyl cis/trans isomerases (PPIases), comprising cylcophilins and FK506-binding proteins (FKBPs), the specific target of these drugs, were identified in the Plasmodium parasite and proposed as an antimalarial drug target. We previously attempted to decipher the structure of these proteins and target them with non-immunosuppressive drugs, predominantly on FKBP35. This review summarizes the structural insights on Plasmodium PPIases, their inhibitor complexes and perspectives on drug discovery.

Prostaglandin A2 Interacts with Nurr1 and Ameliorates Behavioral Deficits in Parkinson's Disease Fly Model.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance, and protection of midbrain dopaminergic neurons. Recently, we demonstrated that prostaglandins E1 (PGE1) and PGA1 directly bind to the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional activation function. In this direction, here we report the transcriptional activation of Nurr1 by PGA2, a dehydrated metabolite of PGE2, through physical binding ably supported by NMR titration and crystal structure. The co-crystal structure of Nurr1-LBD bound to PGA2 revealed the covalent coupling of PGA2 with Nurr1-LBD through Cys566. PGA2 binding also induces a 21° shift of the activation function 2 (AF-2) helix H12 away from the protein core, similar to that observed in the Nurr1-LBD-PGA1 complex. We also show that PGA2 can rescue the locomotor deficits and neuronal degeneration in LRRK2 G2019S transgenic fly models.

Antiviral activity against Middle East Respiratory Syndrome coronavirus by Montelukast, an anti-asthma drug.

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections.

Special Issue "Selected Papers from the 8th Asia-Pacific NMR (APNMR) Symposium: Recent Advances in NMR Spectroscopy".

Asia-Pacific NMR (APNMR) has been an important scientific event in the region, engaging a large number of NMR scientists from academia and industries [...].

PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson's disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1's transcriptional function.

Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases.

For over a half-century the anti-malarial drug chloroquine (CQ) has been used as a therapeutic agent, alone or in combination, to treat autoimmune diseases. However, neither the underlying mechanism(s) of action nor their molecular target(s) are well defined. The orphan nuclear receptor Nurr1 (also known as NR4A2) is an essential transcription factor affecting the development and maintenance of midbrain dopaminergic neurons. In this study, using in vitro T cell differentiation models, we demonstrate that CQ activates TREG cell differentiation and induces Foxp3 gene expression in a Nurr1-dependent manner. Remarkably, CQ appears to induce Nurr1 function by two distinct mechanisms: firstly, by direct binding to Nurr1's ligand-binding domain and promoting its transcriptional activity and secondly by upregulation of Nurr1 expression through the CREB signaling pathway. In contrast, CQ suppressed gene expression and differentiation of pathogenic TH17 cells. Importantly, using a valid animal model of inflammatory bowel disease (IBD), we demonstrated that CQ promotes Foxp3 expression and differentiation of TREG cells in a Nurr1-dependent manner, leading to significant improvement of IBD-related symptoms. Taken together, these data suggest that CQ ameliorates autoimmune diseases via regulating Nurr1 function/expression and that Nurr1 is a promising target for developing effective therapeutics of human inflammatory autoimmune diseases.

Crystal structure of human vaccinia-related kinase 1 in complex with AMP-PNP, a non-hydrolyzable ATP analog.

Vaccinia-related kinase 1 (VRK1), a serine/threonine mitotic kinase, is widely over-expressed in dividing cells and regarded as a cancer drug target primarily due to its function as an early response gene in cell proliferation. However, the mechanism of VRK1 phosphorylation and substrate activation is not well understood. More importantly even the molecular basis of VRK1 interaction with its cofactor, adenosine triphosphate (ATP), is unavailable to-date. As designing specific inhibitors remains to be the major challenge in kinase research, such a molecular understanding will enable us to design ATP-competitive specific inhibitors of VRK1. Here we report the molecular characterization of VRK1 in complex with AMP-PNP, a non-hydrolyzable ATP-analog, using NMR titration followed by the co-crystal structure determined upto 2.07 Å resolution. We also carried out the structural comparison of the AMP-PNP bound-form with its apo and inhibitor-bound counterparts, which has enabled us to present our rationale toward designing VRK1-specific inhibitors.

Functionalized MoS2 Nanosheets as Multi-Gene Delivery Vehicles for In Vivo Pancreatic Cancer Therapy.

Transition metal dichalcogenides (TMDCs) are categorized as novel two-dimensional (2D) nanomaterials with unique physical and chemical properties, bearing varied applications in medical and materials sciences. However, only a few works report the application of TMDCs for gene therapy in cancer treatment. Here, we engineer a multi-gene delivery system based on functionalized monolayer MoS2, which can co-deliver HDAC1 and KRAS small interfering RNAs (siRNAs) to Panc-1 cancer cells for combinational cancer therapy. The synergistic effect of gene silencing therapy and NIR phototherapy is demonstrated by inhibition of both genes, in vitro cell growth rate, and in vivo tumor volume growth rate, exemplifying pre-eminent anticancer efficacy. This anti-tumor effect is a result of the photothermal effect of MoS2 induced by NIR excitation and inactivation of HDAC1 and KRAS genes, which consequently bring about apoptosis, inhibit migration, and induce cell cycle arrest in the treated Panc-1 cells. Moreover, good biocompatibility and reduced cytotoxicity of MoS2-based nanocarriers enable their metabolism within in vitro and in vivo mouse models over a prolonged duration without any evident ill-effects. In summary, we demonstrate the promising potential of low-toxicity, functionalized MoS2 nanocarriers as a biocompatible gene delivery system for in vivo pancreatic adenocarcinoma therapy.

Molecular diversity and function of jasmintides from Jasminum sambac.

BACKGROUND: Jasmintides jS1 and jS2 from Jasminum sambac were previously identified as a novel family of cysteine-rich peptides (CRPs) with an unusual disulfide connectivity. However, very little else is known about jasmintides, particularly their molecular diversity and functions. Here, we report the discovery and characterization of a novel suite of jasmintides from J. sambac using transcriptomic, peptidomic, structural and functional tools. RESULTS: Transcriptomic analysis of leaves, flowers and roots revealed 14 unique jasmintide precursors, all of which possess a three-domain architecture comprising a signal peptide, a pro-domain and a mature jasmintide domain. Peptidomic analysis, using fractionated mixtures of jasmintides and chemical derivatization of cysteine to pseudolysine, trypsin digestion and MS/MS sequencing, revealed an additional 86 jasmintides, some of which were post-translationally modified. NMR analysis showed that jasmintide jS3 has three anti-parallel ß-strands with a three-disulfide connectivity of CysI-CysV, CysII-CysIV and CysIII-CysVI, which is similar to jasmintide jS1. Jasmintide jS3 was able to withstand thermal, acidic and enzymatic degradation and, importantly, exhibited antifeedant activity against mealworm Tenebrio molitor. CONCLUSION: Together, this study expands the existing library of jasmintides and furthers our understanding of the molecular diversity and cystine framework of CRPs as scaffolds and tools for engineering peptides targeting pests.

Correction: Suprafenacine, an Indazole-Hydrazide Agent, Targets Cancer Cells Through Microtubule Destabilization.

[This corrects the article DOI: 10.1371/journal.pone.0110955.].

Chemogenomic Profiling of Human and Microbial FK506-Binding Proteins.

FK506-binding proteins (FKBPs) are evolutionarily conserved proteins that display peptidyl-prolyl isomerase activities and act as coreceptors for immunosuppressants. Microbial macrophage-infectivity-potentiator (Mip)-type FKBPs can enhance infectivity. However, developing druglike ligands for FKBPs or Mips has proven difficult, and many FKBPs and Mips still lack biologically useful ligands. To explore the scope and potential of C5-substituted [4.3.1]-aza-bicyclic sulfonamides as a broadly applicable class of FKBP inhibitors, we developed a new synthesis method for the bicyclic core scaffold and used it to prepare an FKBP- and Mip-focused library. This allowed us to perform a systematic structure-activity-relationship analysis across key human FKBPs and microbial Mips, yielding highly improved inhibitors for all the FKBPs studied. A cocrystal structure confirmed the molecular-binding mode of the core structure and explained the affinity gained as a result of the preferred substituents. The best FKBP and Mip ligands showed promising antimalarial, antileginonellal, and antichlamydial properties in cellular models of infectivity, suggesting that substituted [4.3.1]-aza-bicyclic sulfonamides could be a novel class of anti-infectives.

Self-association and conformational variation of NS5A domain 1 of hepatitis C virus.

Direct-acting antivirals (DAAs) targeting the non-structural 5A (NS5A) protein of the hepatitis C virus (HCV) are crucial drugs that have shown exceptional clinical success in patients. However, their mode of action (MoA) remains unclear, and drug-resistant HCV strains are rapidly emerging. It is critical to characterize the behaviour of the NS5A protein in solution, which can facilitate the development of new classes of inhibitors or improve the efficacy of the currently available DAAs. Using biophysical methods, including dynamic light scattering, size exclusion chromatography and chemical cross-linking experiments, we showed that the NS5A domain 1 from genotypes 1b and 1a of the HCV intrinsically self-associated and existed as a heterogeneous mixture in solution. Interestingly, the NS5A domain 1 from genotypes 1b and 1a exhibited different dynamic equilibria of monomers to higher-order structures. Using small-angle X-ray scattering, we studied the structural dynamics of the various states of the NS5A domain 1 in solution. We also tested the effect of daclatasvir (DCV), the most prominent DAA, on self-association of the wild and DCV-resistant mutant (Y93H) NS5A domain 1 proteins, and demonstrated that DCV induced the formation of large and irreversible protein aggregates that eventually precipitated out. This study highlights the conformational variability of the NS5A domain 1 of HCV, which may be an intrinsic structural behaviour of the HCV NS5A domain 1 in solution.

Speckle reduction in quantitative phase imaging by generating spatially incoherent laser field at electroactive optical diffusers.

We studied quantitative phase imaging (QPI) using coherent laser illumination coupled with static and moving optical diffusers. The spatial coherence of a continuous-wave laser was controlled by tuning the particle size and the diffusion angle of optical diffusers for speckle-reduced 3D phase imaging of transparent objects. We used a common-path QPI configuration to investigate the coherent phase mapping of polystyrene micro-beads and breast cancer cells (MCF-7) under different degrees of coherent speckles. The proposed speckle reduction method could provide an avenue for enhancing lateral resolution and suppressing coherent artifacts of the phase images from QPI.

Stimuli-responsive multifunctional glyconanoparticle platforms for targeted drug delivery and cancer cell imaging.

Targeted bioimaging or chemotherapeutic drug delivery to achieve the desired therapeutic effects while minimizing side effects has attracted considerable research attention and remains a clinical challenge. Presented herein is a multi-component delivery system based on carbohydrate-functionalized gold nanoparticles conjugated with a fluorophore or prodrug. The system leverages active targeting based on carbohydrate-lectin interactions and release of the payload by biological thiols. Cell-type specific delivery of the activatable fluorophore was examined by confocal imaging on HepG2 cells, and displays distinct selectivity towards HepG2 cells over HeLa and NIH3T3 cells. The system was further developed into a drug delivery vehicle with camptothecin (CPT) as a model drug. It was demonstrated that the complex exhibits similar cytotoxicity to that of free CPT towards HepG2 cells, and is significantly less cytotoxic to normal HDF and NIH3T3 cells, indicating excellent specificity. The delivery vehicle itself exhibits excellent biocompatibility and offers an attractive strategy for cell-type specific delivery depending on the carbohydrates conjugated in the system.

New structural insight of C-terminal region of Syntenin-1, enhancing the molecular dimerization and inhibitory function related on Syndecan-4 signaling.

The PDZ domain-containing scaffold protein, syntenin-1, binds to the transmembrane proteoglycan, syndecan-4, but the molecular mechanism/function of this interaction are unknown. Crystal structure analysis of syntenin-1/syndecan-4 cytoplasmic domains revealed that syntenin-1 forms a symmetrical pair of dimers anchored by a syndecan-4 dimer. The syndecan-4 cytoplasmic domain is a compact intertwined dimer with a symmetrical clamp shape and two antiparallel strands forming a cavity within the dimeric twist. The PDZ2 domain of syntenin-1 forms a direct antiparallel interaction with the syndecan-4 cytoplasmic domain, inhibiting the functions of syndecan-4 such as focal adhesion formation. Moreover, C-terminal region of syntenin-1 reveals an essential role for enhancing the molecular homodimerization. Mutation of key syntenin-1 residues involved in the syndecan-4 interaction or homodimer formation abolishes the inhibitory function of syntenin-1, as does deletion of the homodimerization-related syntenin-1 C-terminal domain. Syntenin-1, but not dimer-formation-incompetent mutants, rescued the syndecan-4-mediated inhibition of migration and pulmonary metastasis by B16F10 cells. Therefore, we conclude that syntenin-1 negatively regulates syndecan-4 function via oligomerization and/or syndecan-4 interaction, impacting cytoskeletal organization and cell migration.

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.

Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition.

Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506.

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25-rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.

Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel ß-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.