Yuji O. Kamatari

Hot spots in prion protein for pathogenic conversion

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrPC) to the scrapie form (PrPSc) is still unknown. Here we discovered a chemical chaperone to stabilize the PrPC conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a “pocket” created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrPSc. Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrPC, will open the way to the development of novel anti-prion drugs.

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNPSc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNPSc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNPSc in addition to attenuation of microgliosis and neuroprotection.

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrPC) of PrP into an abnormal form (PrPSc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrPC is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrPSc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrPC, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.

Exploring the folding energy landscape with pressure.

The unique role of pressure in protein folding studies is emphasized. Variable-pressure NMR experiments carried out under equilibrium conditions give unique opportunities to explore the energy landscape for protein folding. Intermediate conformers that may appear transiently in the kinetic folding experiments may be stably trapped under pressure, allowing examination of their conformations in site-specific detail with modern NMR spectroscopy. The intimate relationship between the kinetic folding experiment and the equilibrium pressure experiment is described with examples from ubiquitin and hen lysozyme.

Cavity hydration as a gateway to unfolding: An NMR study of hen lysozyme at high pressure and low temperature

We have used low temperatures (down to -20°C) and high pressures (up to 2000 bar) to populate low-lying excited state conformers of hen lysozyme, and have analyzed their structures site-specifically using (15)N/(1)H two-dimensional HSQC NMR spectroscopy. The resonances of a number of residues were found to be selectively broadened, as the temperature was lowered at a pressure of 2000 bar. The resulting disappearance of cross-peaks includes those of residues in the β-domain of the protein and the cleft between the β- and α-domains, both located close to water-containing cavities. The results indicate that low-lying excited state conformers of hen lysozyme are characterized by slowly fluctuating local conformations around these cavities, attributed to the opportunities for water molecules to penetrate into the cavities. Furthermore, we have found that these water-containing cavities are conserved in similar positions in lysozymes from a range of different biological species, indicating that they are a common evolutionary feature of this family of enzymes.

Structure-based discovery of anti-influenza virus A compounds among medicines.

BACKGROUND Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme. METHODS We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay. RESULTS We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase. CONCLUSIONS Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase. GENERAL SIGNIFICANCE Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.

Characterizing antiprion compounds based on their binding properties to prion proteins: implications as medical chaperones.

A variety of antiprion compounds have been reported that are effective in ex vivo and in vivo treatment experiments. However, the molecular mechanisms for most of these compounds remain unknown. Here we classified antiprion mechanisms into four categories: I, specific conformational stabilization; II, nonspecific stabilization; III, aggregation; and IV, interaction with molecules other than PrPC. To characterize antiprion compounds based on this classification, we determined their binding affinities to PrPC using surface plasmon resonance and their binding sites on PrPC using NMR spectroscopy. GN8 and GJP49 bound specifically to the hot spot in PrPC, and acted as “medical chaperones” to stabilize the native conformation. Thus, mechanisms I was predominant. In contrast, quinacrine and epigallocathechin bound to PrPC rather nonspecifically; these may stabilize the PrPC conformation nonspecifically including the interference with the intermolecular interaction following mechanism II. Congo red and pentosan polysulfate bound to PrPC and caused aggregation and precipitation of PrPC, thus reducing the effective concentration of prion protein. Thus, mechanism III was appropriate. Finally, CP‐60, an edarabone derivative, did not bind to PrPC. Thus these were classified into mechanism IV. However, their antiprion activities were not confirmed in the GT + FK system, whose details remain to be elucidated. This proposed antiprion mechanisms of diverse antiprion compounds could help to elucidate their antiprion activities and facilitate effective antiprion drug discovery.

Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells.

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC(50) values of 0.51-0.83 μM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.

Molecular analysis of the binding mode of Toll/interleukin-1 receptor (TIR) domain proteins during TLR2 signaling

Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes.

A theoretical study of the two binding modes between lysozyme and tri-NAG with an explicit solvent model based on the fragment molecular orbital method

To examine the stabilities and binding characteristics, fragment molecular orbital (FMO) calculations were performed for the two binding modes of hen egg-white lysozyme with tri-N-acetyl-D-glucosamine (tri-NAG). Solvent effects were considered using an explicit solvent model. For comparison with the computational results, we experimentally determined the enthalpic contribution of the binding free-energy. Our calculations showed that the binding mode observed by X-ray analysis was more stable than the other binding mode by -6.2 kcal mol(-1), where it was found that the interaction of protein with solvent molecules was crucial for this stability. The amplitude of this energy difference was of the same order as the experimental enthalpic contribution. Our detailed analysis using the energies divided into each residue was also consistent with a previous mutant study. In addition, the electron density analysis showed that the formal charge of the lysozyme (+8.0 e) was reduced to +5.16 e by charge transfer with solvent molecules.