Search

Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideβ ( 6 ), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo . We demonstrate that cholicamideβ ( 6 ), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideβ ( 6 ) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideβ ( 6 ) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideβ ( 6 ) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.

Several proteins have been shown to undergo a shift in the mechanism of ligand binding-induced folding from conformational selection (CS; folding precedes binding) to induced fit (IF; binding precedes folding) with increasing ligand concentration. In previous studies of the coupled folding/binding reaction of staphylococcal nuclease (SNase) in the presence of a substrate analogue, adenosine-3',5'-diphosphate (prAp), we found that the two phosphate groups make important energetic contributions toward stabilizing its complex with the native protein as well as transient conformational states encountered at high ligand concentrations favoring IF. However, the structural contributions of each phosphate group during the reaction remain unclear. To address this question, we relied on fluorescence, nuclear magnetic resonance (NMR), absorption, and isothermal titration calorimetry to study the effects of deletion of the phosphate groups of prAp on the kinetics of ligand-induced folding, using a strategy analogous to mutational ϕ-value analysis to interpret the results. Kinetic measurements over a wide range of ligand concentrations, together with structural characterization of a transient protein-ligand encounter complex using 2D NMR, indicated that, at high ligand concentrations favoring IF, (i) the 5'-phosphate group interacts weakly with denatured SNase during early stages of the reaction, resulting in loose docking of the two domains of SNase, and (ii) the 3'-phosphate group engages in some specific contacts with the polypeptide in the transition state prior to formation of the native SNase-prAp complex.

Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ⇔ I ⇔ L ⇔ M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (ΔΔ G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ΔΔ G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ⇔ I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ΔΔ G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.

Supramolecular complexation of γ-cyclodextrin (γ-CD) with N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (DMP-PDI) or N,N'-bis(2,6-dioctyl)perylene-3,4,9,10-tetracarboxylic diimide (C8-PDI) dye in an aqueous solution and in a γ-CD solid film were investigated via ensemble and single-molecule fluorescence spectroscopy. These two perylene diimide derivatives possess almost the same electronic structure but have different terminal functional groups. This structural difference leads to formation of an inclusion complex of γ-CD with DMP-PDI but not with C8-PDI in aqueous solution. In a γ-CD solid film, the distributions of the wavelengths of emission maximum (λ max em ) are strikingly different between these two dyes; a much narrower and blue-shifted λ max em distribution was observed for C8-PDI relative to DMP-PDI. This difference is attributed to the fact that the C8-PDI molecules are bound at the γ-CD/glass interface as a result of spin-coating of the sample solution, whereas the DMP-PDI molecules form 1:1 and 1:2 inclusion complexes with conformational heterogeneities in the film. In comparison to the case for C8-PDI, more frequent on-off blinking events were observed for DMP-PDI. The blinking statistics of DMP-PDI in the γ-CD film exhibit both single-exponential and nonexponential (i.e., dispersive) kinetics, revealed by robust statistical analysis. Energetic consideration with the aid of theoretical calculations suggests that the underlying photophysics most probably involves hydrogen atom transfer (HAT) between the DMP-PDI guest and γ-CD host via higher excited (n, π*) triplet states. The hypothesis of HAT in the inclusion complex reasonably explains the experimental results; however, a charge transfer hypothesis cannot explain the results. The dispersive kinetics is attributable to the effect of thermal fluctuation in the forward and backward HAT reactions.

Despite the major role of pH in protein folding and stability, a quantitative understanding of the pH-induced protein folding mechanism remains elusive. Two conventional models, the Monod-Wyman-Changeux and Linderstrøm-Lang smeared charge models, respectively, have been used to analyze the formation/disruption of specific native structures and fluctuating non-native states. However, there are only a few models that can represent the overall kinetic events of folding/unfolding independent of the properties of relevant molecular species, which has hampered the efforts to systematically analyze pH-induced folding. Here, we constructed a statistical mechanical model that incorporates the protonation mechanism of conventional models along with a combined manual search and least-squares fitting procedure, which was used to investigate the folding of horse apomyoglobin over a wide pH range (2.2-6.7), with a time window ranging from ∼40 μs to ∼100 s, using continuous-/stopped-flow fluorescence at 8 °C. Quantitative analysis assuming a five-state sequential scheme indicated that (1) pH-induced folding/unfolding is represented by both specific binding and Coulombic interactions; (2) kinetic folding/unfolding intermediates share kinetic mechanisms with the equilibrium intermediate, indicating their equivalence; and (3) native-like properties are acquired successively during folding by intermediates and in transition states. This model could also be applied to a variety of association/dissociation processes.

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

Jumonji AT-rich interactive domain 1A (JARID1A), one of the jumonji C domain-containing histone demethylase (JHDM) family members, plays key roles in cancer cell proliferation and development of drug tolerance. Therefore, selective JARID1A inhibitors are potential anticancer agents. In this study, we searched for cell-active JARID1A inhibitors by screening hydroxamate compounds in our in-house library and the structural optimization based on docking study of the hit-compound to a homology model of JARID1A. As a result, we identified compound 6j, which selectively inhibits JARID1A over three other JHDM family members. Compound 7j, a prodrug form of compound 6j, induced a selective increase in the level of trimethylation of histone H3 lysine 4, a substrate of JARID1A. Furthermore, compound 7j synergistically enhanced A549 human lung cancer cell growth inhibition induced by vorinostat, a histone deacetylase inhibitor. These findings support the idea that JARID1A inhibitors have potential as anticancer agents.

Histone N(ε)-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 μM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 μM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

Catalytic asymmetric benzoylation of 1,2-diols has been developed using a solid-phase asymmetric catalyst. The reaction conditions were optimized by the screening of different metal salts, solvents, bases, and temperatures. High-throughput screening was performed using circular dichroism detection, and the results revealed that Nb-imidazoline-copper(I) in combination with diisopropylethylamine was able to catalyze with high enantioselectivity, giving the monobenzoylated products in high yields and excellent enantiomeric excesses of up to 95% ee. [reaction: see text]

To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.

Using low-temperature time-resolved spectroscopy, we have directly observed thermal back reaction of metarhodopsin I (meta I) to lumirhodopsin (lumi) and that of metarhodopsin II (meta II) to meta I in chicken rhodopsin to demonstrate the presence of thermal equilibria among lumi, meta I, and meta II. The back reaction from meta I to lumi was observed when the rhodopsin sample irradiated at -35 degrees C was warmed to -20 degrees C, while that from meta II to meta I was observed when the sample irradiated at -10 degrees C was cooled to -20 degrees C. Thermodynamic parameters of lumi, meta I, and meta II were calculated from the equilibrium constants estimated by analyzing the spectra of the equilibrium states at temperatures ranging from -30 to -10 degrees C. The results showed that meta I has an enthalpy and an entropy considerably smaller than those of lumi and meta II, while the difference in thermodynamic parameters between lumi and meta II is not so large. These results suggest that meta I is a crucial stage of conversion of the light energy captured by the chromophore into restricted conformations of the chromophore and/or protein, from which a large conformational change of the protein starts to form meta II.