Similarity scores of vibrational spectra reveal the atomistic structure of pentapeptides in multiple basins.

Vibrational spectroscopy combined with theoretical calculations is a powerful tool for analyzing the interaction and conformation of peptides at the atomistic level. Nonetheless, identifying the structure becomes increasingly difficult as the peptide size grows large. One example is acetyl-SIVSF-N-methylamide, a capped pentapeptide, whose atomistic structure has remained unknown since its first observation [T. Sekiguchi, M. Tamura, H. Oba, P. Çarçarbal, R. R. Lozada-Garcia, A. Zehnacker-Rentien, G. Grégoire, S. Ishiuchi and M. Fujii, Angew. Chem., Int. Ed., 2018, 57, 5626-5629]. Here, we propose a novel conformational search method, which exploits the structure-spectrum correlation using a similarity score that measures the agreement of theoretical and experimental spectra. Surprisingly, the two conformers have distinctly different energy and geometry. The second conformer is 25 kJ mol-1 higher in energy than the other, lowest-energy conformer. The result implies that there are multiple pathways in the early stage of the folding process: one to the global minimum and the other to a different basin. Once such a structure is established, the second conformer is unlikely to overcome the barrier to produce the most stable structure due to a vastly different hydrogen bond network of the backbone. Our proposed method can characterize the lowest-energy conformer and kinetically trapped, high-energy conformers of complex biomolecules.

Conformation-Dependent Influences of Hydrophobic Amino Acids in Two In-Register Parallel ß-Sheet Amyloids, an α-Synuclein Amyloid and a Local Structural Model of PrPSc.

Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc underlies the existence of many prion strains and species barriers of prions, although the conformational information is extremely limited. Interestingly, differences between polymorphic or species-specific residues responsible for the species/strain barriers are often caused by conservative replacements between hydrophobic amino acids. This implies that subtle differences among hydrophobic amino acids are significant for PrPSc structures. Here we analyzed the influence of different hydrophobic residues on the structures of an in-register parallel ß-sheet amyloid of α-synuclein (αSyn) using molecular dynamics (MD) simulation and applied the knowledge from the αSyn amyloid to modeling a local structure of human PrPSc encompassing residues 107-143. We found that mutations equivalent to polymorphisms that cause transmission barriers substantially affect the stabilities of the local structures; for example, the G127V mutation, which makes the host resistant to various human prion diseases, greatly destabilized the local structure of the model amyloid. Our study indicates that subtle differences among hydrophobic side chains can considerably affect the interaction network, including hydrogen bonds, and demonstrates specifically how and in what structures hydrophobic residues can exert unique effects on in-register parallel ß-sheet amyloids.

Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

Systemic lupus erythematosus overlapping dermatomyositis owing to a heterozygous TREX1 Asp130Asn missense mutation.

The 3' repair exonuclease 1 (TREX1) gene encodes a nuclear protein with 3' exonuclease activity, and the mutations have been associated with autoimmune diseases. Herein, we performed genetic analysis for the TREX1 gene in 55 patients with systemic lupus erythematosus (SLE). We identified one SLE patient with overlapping dermatomyositis having a heterozygous p.Asp130Asn mutation in the TREX1 gene. The patient had a high level of serum interferon (IFN)-α compared with that in healthy controls and other patients with SLE. In addition, the patient expressed elevated IFN signature genes compared with healthy controls. Our molecular dynamics simulation of the TREX1 protein in a complex with double-stranded DNA revealed that the D130N mutant causes significant changes in the active site's interaction network. One of our cases exhibited a heterozygous TREX1 p.Asp130Asn mutation that contributed to the type I IFN pathway, which may lead to the development of a severe SLE phenotype.

Idiopathic multicentric Castleman disease with novel heterozygous Ile729Met mutation in exon 10 of familial Mediterranean fever gene.

OBJECTIVE: While the aetiology of idiopathic multicentric Castleman disease (iMCD) remains unclear, the involvement of autoinflammatory mechanisms has been suggested. Herein we report a Japanese patient with iMCD with a novel heterozygous Ile729Met mutation in exon 10 of the Mediterranean fever (MEFV) gene. METHODS: We performed genetic analysis via targeted next-generation sequencing analysis and Sanger sequencing and conducted molecular dynamics simulations to investigate the hydrophobic interactions around the 729th amino acid in human pyrin. RESULTS: In February 2011, a 59-year-old man was diagnosed with IgG4-related disease at our department based on the findings of cervical and hilar lymphadenopathies, typical lung lesions and cervical lymph node biopsy. The patient was followed up without treatment, as he was asymptomatic. However, he had been experiencing prolonged fatigue and fever with high levels of CRP since June 2017. Axillary lymph node biopsy findings led to the diagnosis of iMCD. He was successfully treated with an IL-6 inhibitor and has been in remission for 12 months. Genetic analyses for hereditary autoinflammatory disease-related genes were performed, revealing a novel heterozygous Ile729Met mutation in exon 10 of the MEFV gene. We identified that this novel mutation significantly altered the local interaction of the human pyrin B30.2 domain by molecular dynamics simulation analysis and experimentally had the potential for inflammasome activation with increased inflammatory cytokines. CONCLUSION: The abnormal function of pyrin due to a mutation in the MEFV gene in this patient may have contributed to the development of MCD by inducing IL-6 production via inflammasome signalling.

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants.

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs.

Mechanisms of Strain Diversity of Disease-Associated in-Register Parallel ß-Sheet Amyloids and Implications About Prion Strains.

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel ß-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel ß-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between ß-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.

Synthesis of Trifluoromethyl-α,ß-unsaturated Lactones and Pyrazolinones and Discovery of Influenza Virus Polymerase Inhibitors.

To explore the potential biological activities of trifluoromethyl heterocycles, we recently developed a synthetic approach to access a series of α-trifluoromethyl-α,ß-unsaturated lactones and trifluoromethyl pyrazolinones. The compounds were tested for their antimicrobial activity, and we found that some compounds had anti-influenza viral activity. The ß-aryl-α-trifluoromethyl α,ß-unsaturated lactone derivatives 5 g (5-(4-chlorophenyl)-5-methyl-4-phenyl-3-(trifluoromethyl)furan-2-one), 7 b (4-(4-methoxyphenyl)-3-(trifluoromethyl)spiro[furan-5,1'-indane]-2-one), and the trifluoromethyl pyrazolinone 12 c (1-(6-methoxy-2-naphthyl)-2-(trifluoromethyl)-5,6,7,8-tetrahydropyrazolo[1,2-a]pyridazin-3-one) were found to possess promising inhibitory activity against influenza virus type A, strain A/WSN/33 (H1N1). These three hit compounds were successfully optimized, and we identified that the most potent compound 5 h (5-(4-chlorophenyl)-4-(6-methoxy-2-naphthyl)-5-methyl-3-(trifluoromethyl)furan-2-one) showed inhibitory activity against various types of influenza A and B viruses in the low-micromolar range without showing cytotoxicity. Moreover, 5 h was more effective against the clinical isolate A/California/7/2009 (H1N1pdm) strain than the influenza viral polymerase inhibitor, favipiravir (T-705). We also delineated the structure-activity relationship and obtained mechanistic insight into inhibition of the viral polymerase.

Disulfide-crosslink scanning reveals prion-induced conformational changes and prion strain-specific structures of the pathological prion protein PrPSc.

Prions are composed solely of the pathological isoform (PrPSc) of the normal cellular prion protein (PrPC). Identification of different PrPSc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrPSc However, these structures remain yet to be identified, because of the incompatibility of PrPSc with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1∼H2) of PrPC might cooperate with the more C-terminal side region for efficient interactions with PrPSc From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide-crosslink between H1∼H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, Cys substitutions at residues 165, 166, or 168 in H1∼H2 were combined with cysteine scanning along Ctrm residues 220-229. We found that C;C-PrPs are expressed normally with glycosylation patterns and subcellular localization similar to WT PrP, albeit differing in expression levels. Interestingly, some C;C-PrPs converted to protease-resistant isoforms in the 22L-ScN2a cells, but not in Fukuoka1 prion-infected cells. Crosslink patterns of convertible C;C-PrPs indicated a positional change of H1∼H2 toward Ctrm in PrPSc-induced conformational conversion. Given the properties of the C;C-PrPs reported here, we propose that these PrP variants may be useful tools for investigating prion strain-specific structures and structure-phenotype relationships of PrPSc.

Structure-based drug discovery for combating influenza virus by targeting the PA-PB1 interaction.

Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the currently-approved anti-influenza viral drugs. We performed in silico screening using a structure-based drug discovery algorithm called Nagasaki University Docking Engine (NUDE), which is optimised for a GPU-based supercomputer (DEstination for Gpu Intensive MAchine; DEGIMA), by targeting influenza viral PA protein. The compounds selected by NUDE were tested for anti-influenza virus activity using a cell-based assay. The most potent compound, designated as PA-49, is a medium-sized quinolinone derivative bearing a tetrazole moiety, and it inhibited the replication of influenza virus A/WSN/33 at a half maximal inhibitory concentration of 0.47 µM. PA-49 has the ability to bind PA and its anti-influenza activity was promising against various influenza strains, including a clinical isolate of A(H1N1)pdm09 and type B viruses. The docking simulation suggested that PA-49 interrupts the PA-PB1 interface where important amino acids are mostly conserved in the virus strains tested, suggesting the strain independent utility. Because our NUDE/DEGIMA system is rapid and efficient, it may help effective drug discovery against the influenza virus and other emerging viruses.

Computational study of the competitive binding of valproic acid glucuronide and carbapenem antibiotics to acylpeptide hydrolase.

The efficacy of the antiepileptic drug VPA is decreased by co-administered carbapenems (CBPMs). The mechanism of CBPM selective inhibition of acylpeptide hydrolase (APEH) hydrolysis of VPA-glucuronide (VPA-G) to VPA is unclear due to the lack of APEH structural information. Here we performed homology modeling of the three-dimensional structure of APEH and subsequent docking simulations with a modeled structure to understand this mechanism. Docking simulations indicated that four groups of binding structures were involved in the binding of VPA-G, panipenem, and meropenem to APEH, but only one or two binding structures were involved in the binding of meropenem with an open ß-lactam ring structure and other antibiotics involving ampicillin. One of the four VPA-G binding structures was close enough to the APEH catalytic triad to facilitate VPA-G hydrolysis. This binding structure was also the most stable binding structure for panipenem, suggesting potential inhibition of VPA-G hydrolysis by panipenem. Fragment molecular orbital calculations of interaction energies of amino acid residues of APEH with VPA-G, panipenem, and meropenem indicated that the binding structure for panipenem closest to the catalytic triad is stabilized upon APEH interaction. These data suggest that APEH binding characteristics with CBPMs may help explain the selective inhibition of APEH by CBPMs.

Ionic Liquid-Mediated Hydrofluorination of o-Azaxylylenes Derived from 3-Bromooxindoles.

The hydrofluorination reaction of 3-bromooxindole using mild HF reagents in an ionic liquid is described. This transformation can operate at room temperature to give a series of 3-substituted 3-fluorooxindole derivatives including racemic BMS 204352 (MaxiPost). The mechanistic study about interactions between HF and 3-butyl-1-methylimidazolium tetrafluoroborate [bmim][BF4] is also discussed on the basis of energy calculations.

Anharmonic Vibrational Analyses of Pentapeptide Conformations Explored with Enhanced Sampling Simulations.

An accurate theoretical prediction of the vibrational spectrum of polypeptides remains to be a challenge due to (1) their conformational flexibility and (2) non-negligible anharmonic effects. The former makes the search for conformers that contribute to the spectrum difficult, and the latter requires an expensive, quantum mechanical calculation for both electrons and vibrations. Here, we propose a new theoretical approach, which implements an enhanced conformational sampling by the replica-exchange molecular dynamics method, a structural clustering to identify distinct conformations, and a vibrational structure calculation by the second-order vibrational quasi-degenerate perturbation theory (VQDPT2). A systematic mode-selection scheme is developed to reduce the cost of VQDPT2 and the generation of a potential energy surface by the electronic structure calculation. The proposed method is applied to a pentapeptide, SIVSF-NH2, for which the infrared spectrum has recently been measured in the gas phase with high resolution in the OH and NH stretching region. The theoretical spectrum of the lowest energy conformer is obtained with a mean absolute deviation of 11.2 cm-1 from the experimental spectrum. Furthermore, the NH stretching frequencies of the five lowest energy conformers are found to be consistent with the literature values measured for small peptides with a similar secondary structure. Therefore, the proposed method is a promising way to analyze the vibrational spectrum of polypeptides.

Phylogenetic inference of the porcine Rotavirus A origin of the human G1 VP7 gene.

Rotavirus A (RVA) is an important cause of acute gastroenteritis in children worldwide. The most common VP7 genotype of human RVA is G1, but G1 is rarely detected in porcine strains. To understand the evolutionary relationships between human and porcine G1 VP7 genes, we sequenced the VP7 genes of three Japanese G1 porcine strains; the first two (PRV2, S80B) were isolated in 1980 and the third (Kyusyu-14) was isolated in 2001. Then, we performed phylogenetic and in-silico structural analyses. All three VP7 sequences clustered into lineage VI, and the mean nucleotide sequence identity between any pair of porcine G1 VP7 sequences belonging to lineage VI was 91.9%. In contrast, the mean nucleotide sequence identity between any pair of human G1 VP7 sequences belonging to lineages I-V was 95.5%. While the mean nucleotide sequence identity between any pair of porcine lineage VI strain and human lineage I-V strain was 85.4%, the VP7 genes of PRV2 and a rare porcine-like human G1P[6] strain (AU19) were 98% identical, strengthening the porcine RVA origin of AU19. The phylogenetic tree suggests that human G1 VP7 genes originated from porcine G1 VP7 genes. The time of their most recent common ancestor was estimated to be 1948, and human and porcine RVA strains evolved along independent pathways. In-silico structural analyses identified 7 amino acid residues within the known neutralisation epitopes that show differences in electric charges and shape between different porcine and human G1 strains. When compared with much divergent porcine G1 VP7 lineages, monophyletic, less divergent human G1 VP7 lineages support the hypothesis that all human G1 VP7 genes included in this study originated from a rare event of a porcine RVA transmitting to humans that was followed by successful adaptation to the human host. By contrast, AU19 represents interspecies transmission that terminated in dead-end infection.

Vibrational quasi-degenerate perturbation theory with optimized coordinates: applications to ethylene and trans-1,3-butadiene.

A perturbative extension to optimized coordinate vibrational self-consistent field (oc-VSCF) is proposed based on the quasi-degenerate perturbation theory (QDPT). A scheme to construct the degenerate space (P space) is developed, which incorporates degenerate configurations and alleviates the divergence of perturbative expansion due to localized coordinates in oc-VSCF (e.g., local O-H stretching modes of water). An efficient configuration selection scheme is also implemented, which screens out the Hamiltonian matrix element between the P space configuration (p) and the complementary Q space configuration (q) based on a difference in their quantum numbers (λpq = ∑s|ps - qs|). It is demonstrated that the second-order vibrational QDPT based on optimized coordinates (oc-VQDPT2) smoothly converges with respect to the order of the mode coupling, and outperforms the conventional one based on normal coordinates. Furthermore, an improved, fast algorithm is developed for optimizing the coordinates. First, the minimization of the VSCF energy is conducted in a restricted parameter space, in which only a portion of pairs of coordinates is selectively transformed. A rational index is devised for this purpose, which identifies the important coordinate pairs to mix from others that may remain unchanged based on the magnitude of harmonic coupling induced by the transformation. Second, a cubic force field (CFF) is employed in place of a quartic force field, which bypasses intensive procedures that arise due to the presence of the fourth-order force constants. It is found that oc-VSCF based on CFF together with the pair selection scheme yields the coordinates similar in character to the conventional ones such that the final vibrational energy is affected very little while gaining an order of magnitude acceleration. The proposed method is applied to ethylene and trans-1,3-butadiene. An accurate, multi-resolution potential, which combines the MP2 and coupled-cluster with singles, doubles, and perturbative triples level of electronic structure theory, is generated and employed in the oc-VQDPT2 calculation to obtain the fundamental tones as well as selected overtones/combination tones coupled to the fundamentals through the Fermi resonance. The calculated frequencies of ethylene and trans-1,3-butadiene are found to be in excellent agreement with the experimental values with a mean absolute error of 8 and 9 cm(-1), respectively.

The role of intermolecular hydrogen bond on dielectric properties in hydrogen-bonded material 5-bromo-9-hydroxyphenalenone: theoretical investigation.

Dielectric properties of the hydrogen-bonded material, 5-bromo-9-hydroxyphenalenone (C(13)H(7)O(2)Br; BrHPLN), are investigated theoretically by means of electronic structure calculations and Monte Carlo simulations. The density functional calculations of BrHPLN crystals have revealed that the polarization per one molecule can be about 1.7 times larger than that of the isolated monomer. It is also found that there exists significant electron density (0.01 e bohr(-3)) in an intermolecular C-H···O region, which, together with the interatomic distances of 2.39 Å for H···O and 3.34 Å for C···O, suggests the existence of intermolecular weak hydrogen bonding that may enhance the molecular polarization. The induced polarization effects in various intermolecular configurations are evaluated with the Fragment Molecular Orbital method. In addition to the π-π stacking interactions, two types of "in plane" intermolecular weak hydrogen-bonding configurations are found to affect the molecular dipole moment most significantly. These effects are efficiently included in a Monte Carlo simulation method in terms of "dipole corrections" as functions of both the intermolecular arrangements and the intramolecular proton configurations. The application to the dielectric phase transition of a BrHPLN crystal shows that the dipole corrections almost double the transition temperature, toward better agreement with experiments, and qualitatively affect the temperature dependence of the dielectric constant. Discussions are given to support that the results will remain adequate and consistent even after explicit inclusion of the quantum tunneling effects.