Exploring Enzyme Encapsulation Efficiency in MOFs Using Eco-friendly Approaches.

The encapsulation of protein enzymes in metal-organic frameworks (MOFs) has been recognized as an effective enzyme immobilization approach. In this study, we demonstrated the influence of enzyme amount and the isoelectric points (pI) of different enzymes on the enzyme loading capacity in both mechanochemical (ball-milling) and water-based approaches. We found that increasing enzyme amounts enhances MOF enzyme loading without compromising activity, while the MOF shell protects encapsulated enzymes from proteinase K degradation through its size-sheltering mechanism. However, an excess of enzymes can hinder the formation of ZIF-90. Moreover, enzymes with low pI values (e.g., catalase, pI 5.4) facilitate encapsulation in MOFs, whereas enzymes with high pI values (e.g., lysozyme, pI 11.35) are more challenging to encapsulate. The simulation results revealed that increasing the enzyme amounts and pI values raises the activation energy necessary for MOF formation. This study highlights the crucial role of enzyme properties in the encapsulation process within MOFs, providing valuable insights for fabricating enzyme-MOF biocomposites for diverse applications, such as protein drug delivery.

Decoding the Biomimetic Mineralization of Metal-Organic Frameworks in Water.

This study unveils the "green" metal-organic framework (MOF) structuring mechanism by decoding proton transfer in water during ZIF-8 synthesis. Combining in situ small- to wide-angle X-ray scattering, multiscale simulations, and quantum calculations, we reveal that the ZIF-8 early-stage nucleation and crystallization process in aqueous solution unfolds in three distinct stages. In stage I, imidazole ligands replace water in zinc-water cages, triggering an "acidity flip" that promotes proton transfer. This leads to the assembly of structures from single zinc ions to 3D amorphous cluster nuclei. In stage II, amorphous nuclei undergo a critical transformation, evolving into crystalline nuclei and subsequently forming mesoscale-ordered structures and crystallites. The process proceeds until the amorphous precursors are completely consumed, with the transformation kinetics governed by an energy barrier that determines the rate-limiting step. In stage III, stable crystallite nanoparticles form in solution, characterized by a temperature-dependent thermal equilibrium of molecular interactions at the crystal-solution interface. Beyond these core advancements, we explore the influence of encapsulated pepsin and nonencapsulated lysozyme on ZIF-8 formation, finding that their amino acid proton transfer capacity and concentration influence the resulting biomolecule-MOF composite's shape and encapsulation efficiency. The findings contribute to understanding the molecular mechanisms behind biomimetic mineralization and have potential implications for engineering proteins within amorphous MOF nuclei as protein embryo growth sites.

Light Induced Proton Coupled Charge Transfer Triggers Counterion Directional Translocation.

We demonstrate directed translocation of ClO4 - anions from cationic to neutral binding site along the synthetized BPym-OH dye molecule that exhibits coupled excited-state intramolecular proton-transfer (ESIPT) and charge-transfer (CT) reaction (PCCT). The results of steady-state and time-resolved spectroscopy together with computer simulation and modeling show that in low polar toluene the excited-state redistribution of electronic charge enhanced by ESIPT generates the driving force, which is much stronger than by CT reaction itself and provides more informative gigantic shifts of fluorescence spectra signaling on ultrafast ion motion. The associated with ion translocation red-shifted fluorescence band (at 750 nm, extending to near-IR region) appears at the time ~83 ps as a result of electrochromic modulation of PCCT reaction. It occurs at substantial delay to PCCT that displayed fluorescence band at 640 nm and risetime of <200 fs. Thus, it becomes possible to visualize the manifestations of light-triggered ion translocation and of its driving force by fluorescence techniques and to separate them in time and energy domains.

Arrayed Pt Single Atoms via Phosphotungstic Acids Intercalated in Silicate Nanochannels for Efficient Hydrogen Evolution Reactions.

Single-atom catalysts, known for their high activity, have garnered significant interest. Currently, single-atom catalysts were prepared mainly on 2D substrates with random distribution. Here, we report a strategy for preparing arrayed single Pt (Pt1) atoms, which are templated through coordination with phosphotungstic acids (PTA) intercalated inside hexagonally packed silicate nanochannels for a high single Pt-atom loading of ca. 3.0 wt %. X-ray absorption spectroscopy, high-angle annular dark-field scanning transmission electron microscopy, and energy-dispersive X-ray spectroscopy, in conjunction with the density-functional theory calculation, collectively indicate that the Pt single atoms are stabilized via a four-oxygen coordination on the PTA within the nanochannels' inner walls. The critical reduction in the Pt-adsorption energy to nearly the cohesive energy of Pt clustering is attributed to the interaction between PTA and the silicate substrate. Consequently, the transition from single-atom dispersion to clustering of Pt atoms can be controlled by adjusting the number density of PTA intercalated within the silicate nanochannels, specifically when the number ratio of Pt atoms to PTA changes from 3.7 to 18. The 3D organized Pt1-PTA pairs, facilitated by the arrayed silicate nanochannels, demonstrate high and stable efficiency with a hydrogen production rate of ca. 300 mmol/h/gPt─approximately twice that of the best-reported Pt efficiency in polyoxometalate-based photocatalytic systems.

An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation.

The structure determination of protein tyrosine phosphatase (PTP): phospho-protein complexes, which is essential to understand how specificity is achieved at the amino acid level, remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we have established an integrative workflow to address this problem, by means of which we generate a protein:phospho-protein complex model using predetermined protein structures, SAXS and pTyr-tailored MD simulations. Our model reveals transient protein-protein interactions between rPTPεD1 and phospho-SrcKD and is supported by three independent experimental validations. Measurements of the association rate between rPTPεD1 and phospho-SrcKD showed that mutations on the rPTPεD1: SrcKD complex interface disrupts these transient interactions, resulting in a reduction in protein-protein association rate and, eventually, phosphatase activity. This integrative approach is applicable to other PTP: phospho-protein complexes and the characterization of transient protein-protein interface interactions.

Long-range hydrogen-bond relay catalyses the excited-state proton transfer reaction.

Solvent (e.g., water)-catalyzed proton transfer (SCPT) via the relay of hydrogen (H)-bonds plays a key role in proton migration. In this study, a new class of 1H-pyrrolo[3,2-g]quinolines (PyrQs) and their derivatives were synthesized, with sufficient separation of the pyrrolic proton donating and pyridinic proton accepting sites to probe excited-state SCPT. There was dual fluorescence for all PyrQs in methanol, i.e., normal (PyrQ) and tautomer 8H-pyrrolo[3,2-g]quinoline (8H-PyrQ) emissions. The fluorescence dynamics unveiled a precursor (PyrQ) and successor (8H-PyrQ) relationship and the correlation of an increasing overall excited-state SCPT rate (kSCPT) upon increasing the N(8)-site basicity. kSCPT can be expressed by the coupling reaction kSCPT = Keq × kPT, where kPT denotes the intrinsic proton tunneling rate in the relay and Keq denotes the pre-equilibrium between randomly and cyclically H-bonded solvated PyrQs. Molecular dynamics (MD) simulation defined the cyclic PyrQs and analyzed the H-bond and molecular arrangement over time, which showed the cyclic PyrQs incorporating ≧3 methanol molecules. These cyclic H-bonded PyrQs are endowed with a relay-like proton transfer rate, kPT. MD simulation estimated an upper-limited Keq value of 0.02-0.03 for all studied PyrQs. When there was little change in Keq, the distinct kSCPT values for PyrQs were at different kPT values, which increased as the N(8) basicity increased, which was induced by the C(3)-substituent. kSCPT was subject to a deuterium isotope effect, where the kSCPT of 1.35 × 1010 s-1 for PyrQ-D in CH3OD was 1.68 times slower than that (2.27 × 1010 s-1) of PyrQ in CH3OH. MD simulation provided a similar Keq for PyrQ and PyrQ-D, leading to different proton tunneling rates (kPT) between PyrQ and PyrQ-D.

Calculation of CYP450 protein-ligand binding and dissociation free energy paths.

The function of an enzyme depends on its dynamic structure, and the catalytic mechanism has long been an active focus of research. The principle for interpreting protein selectivity and fidelity stems from optimization of the active site upon protein-substrate complexation, i.e., a lock-and-key configuration, on which most protein-substrate molecule binding recognition, and hence drug discovery, relies. Yet another thought has been to incorporate the protein folding interior tunnels for stereo- and regio-selectivity along the protein-substrate or protein-ligand/inhibitor binding process. Free energy calculations provide valuable information for molecular recognition and protein-ligand binding dynamics and kinetics. In this study, we focused on the kinetics of cytochrome P450 proteins (CYP450s) and the protein interior tunnel structure-dynamics relationship in terms of the substrate binding and leaving mechanism. A case in point is given by the prostaglandin H2 (PGH2) homologous isomerase of prostacyclin synthase. To calculate the reactant and product traversing the tunnels to and from the heme site, the free energy paths and tunnel potentials of mean force are constructed from steered molecular dynamics simulations and adaptive basing force umbrella sampling simulations. We explore the binding tunnels and critical residue lining characteristics for the ligand traverse and the underlying mechanism of CYP450 activity. Our theoretical analysis provides insights into the decisive role of the substrate tunnel binding process of the CYP450 mechanism and may be useful in drug design and protein engineering contexts.

Temperature effect on water dynamics in tetramer phosphofructokinase matrix and the super-arrhenius respiration rate.

Advances in understanding the temperature effect on water dynamics in cellular respiration are important for the modeling of integrated energy processes and metabolic rates. For more than half a century, experimental studies have contributed to the understanding of the catalytic role of water in respiration combustion, yet the detailed water dynamics remains elusive. We combine a super-Arrhenius model that links the temperature-dependent exponential growth rate of a population of plant cells to respiration, and an experiment on isotope labeled 18O2 uptake to H218O transport role and to a rate-limiting step of cellular respiration. We use Phosphofructokinase (PFK-1) as a prototype because this enzyme is known to be a pacemaker (a rate-limiting enzyme) in the glycolysis process of respiration. The characterization shows that PFK-1 water matrix dynamics are crucial for examining how respiration (PFK-1 tetramer complex breathing) rates respond to temperature change through a water and nano-channel network created by the enzyme folding surfaces, at both short and long (evolutionary) timescales. We not only reveal the nano-channel water network of PFK-1 tetramer hydration topography but also clarify how temperature drives the underlying respiration rates by mapping the channels of water diffusion with distinct dynamics in space and time. The results show that the PFK-1 assembly tetramer possesses a sustainable capacity in the regulation of the water network toward metabolic rates. The implications and limitations of the reciprocal-activation-reciprocal-temperature relationship for interpreting PFK-1 tetramer mechanisms are briefly discussed.

Could Chemical Reaction at the Molecular Level Show Distinction between Two Liquid-Water States? Study of the Excited-State Water-Catalyzed Proton Transfer Reaction Provides a Clue.

The two liquid-water states, which lead to some anomalies when temperature crosses over 50 ± 10 °C at the atmospheric pressure, have been continuously catching popular attention. In this study, using the excited-state proton transfer (ESPT) catalyzed by water molecules as a prototypical reaction, we demonstrate that the kinetics of ESPT indeed is influenced by the two liquid-water states. In the water-catalyzed ESPT of 3-cyano-7-azaindole (3CAI), a repetitive and comprehensive temperature-dependent study of ESPT in H2O from 0 to 90 °C shows anomalous behavior. The plot of the logarithm of ESPT rate constant as a function of inverse of absolute temperature deviates from a straight line. The convex-Arrhenius behavior manifests the activation free energy for water-assisted ESPT being dependent on temperature and hence the liquid water structure. To simplify the discussion, the plot is well fitted by using two straight lines that are crossed over in the vicinity of 40 °C. The free energy difference between water-solvated 3CAI and the 1:1 H2O:3CAI complex is deduced to be 2.29 ± 0.04 and 1.96 ± 0.04 kcal·mol-1 in the regions of 0-40 and 40-90 °C water, respectively, which also results in different frequency factors, i.e., the proton transfer/tunneling rates of (5.83 ± 0.36) × 1010 and (3.48 ± 0.27) × 1010 s-1, respectively. In a qualitative manner, the results are then rationalized by the different types of H-bonding configuration as proposed for two liquid-water phases, rendering experimental evidence to support the different water phases in ambient temperatures at 1 bar.

TCD, a triterpenoid isolated from wild bitter gourd, reduces synaptosomal release of glutamate and protects against kainic acid-induced neuronal death.

3ß,7ß,25-Trihydroxycucurbita-5,23(E)-dien-19-al (TCD) is a triterpenoid isolated from wild bitter gourd that is a common tropical vegetable with neuroprotective effects. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the aims of this study were to examine the effect of TCD on glutamate release in vitro and to examine the effect of TCD in vivo. In rat cerebrocortical synaptosomes, TCD reduced 4-aminopyridine (4-AP)-stimulated glutamate release and Ca2+ concentration elevation, but had no effect on plasma membrane potential. TCD-mediated inhibition of 4-AP-induced glutamate release was dependent on the presence of extracellular calcium; persisted in the presence of the glutamate transporter inhibitor dl-TBOA, P/Q-type Ca2+ channel blocker ω-agatoxin IVA, and intracellular Ca2+-releasing inhibitors dantrolene and CGP37157; and was blocked by the vesicular transporter inhibitor bafilomycin A1 and the N-type Ca2+ channel blocker ω-conotoxin GVIA. Molecular docking studies have demonstrated that TCD binds to N-type Ca2+ channels. TCD-mediated inhibition of 4-AP-induced glutamate release was abolished by the Ca2+-dependent protein kinase C (PKC) inhibitor Go6976, but was unaffected by the Ca2+-independent PKC inhibitor rottlerin. Furthermore, TCD considerably reduced the phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate, a major presynaptic substrate for PKC. In a rat model of kainic acid (KA)-induced excitotoxicity, TCD pretreatment substantially attenuated KA-induced neuronal death in the CA3 hippocampal region. These results suggest that TCD inhibits synaptosomal glutamate release by suppressing N-type Ca2+ channels and PKC activity and exerts protective effects against KA-induced excitotoxicity in vivo.

[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol, an indole-3-carbinol derivative, inhibits glutamate release in rat cerebrocortical nerve terminals by suppressing the P/Q-type Ca2+ channels and Ca2+/calmodulin/protein kinase A pathway.

Indole-3-carbinol (I3C), found in cruciferous vegetables, has been proposed to exhibit neuroprotective effects. This study aimed to investigate the effect of the I3C derivative [1(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (CIM), which has superior pharmacokinetic properties to I3C, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). We observed that CIM dose-dependently inhibited glutamate release evoked by the potassium channel blocker 4-aminopyridine (4-AP). CIM-mediated inhibition of glutamate release was attributed to reduced exocytosis, as it correlated with the removal of extracellular calcium and blocking of the vesicular glutamate transporter but not the glutamate transporter. In addition, CIM decreased 4-AP-evoked intrasynaptosomal Ca2+ elevation; however, it did not alter the synaptosomal membrane potential. The inhibition of P/Q-typeCa2+ channels abolished the effect of CIM on 4-AP-evoked glutamate release, and the effect was not prevented by intracellular Ca2+ release inhibitors. Moreover, the molecular docking study showed that CIM exhibited the highest binding affinity with the P/Q-type Ca2+channels. Finally, the CIM-mediated inhibition of glutamate release was sensitive to calmodulin, adenylate cyclase (AC), and protein kinase A (PKA) inhibitors. Based on these results, we propose that CIM, through the direct suppression of P/Q-type Ca2+ channels, decreases Ca2+ influx and the activation of Ca2+/calmodulin/AC/PKA signaling, thereby inhibiting glutamate release. This finding is crucial for understanding the role of CIM in the central nervous system and for exploiting its potential in therapeutic interventions.

Probing Interactions between Metal-Organic Frameworks and Freestanding Enzymes in a Hollow Structure.

It has been reported that the biological functions of enzymes could be altered when they are encapsulated in metal-organic frameworks (MOFs) due to the interactions between them. Herein, we probed the interactions of catalase in solid and hollow ZIF-8 microcrystals. The solid sample with confined catalase is prepared through a reported method, and the hollow sample is generated by hollowing the MOF crystals, sealing freestanding enzymes in the central cavities of hollow ZIF-8. During the hollowing process, the samples were monitored by small-angle X-ray scattering (SAXS) spectroscopy, electron microscopy, powder X-ray diffraction (PXRD), and nitrogen sorption. The interfacial interactions of the two samples were studied by infrared (IR) and fluorescence spectroscopy. IR study shows that freestanding catalase has less chemical interaction with ZIF-8 than confined catalase, and a fluorescence study indicates that the freestanding catalase has lower structural confinement. We have then carried out the hydrogen peroxide degradation activities of catalase at different stages and revealed that the freestanding catalase in hollow ZIF-8 has higher activity.

Unveiling the water-associated conformational mobility in the active site of ascorbate peroxidase.

We carried out comprehensive spectroscopic studies of wild type and mutants of ascorbate peroxidase (APX) to gain understanding of the conformational mobility of the active site. In this approach, three unnatural tryptophans were applied to replace the distal tryptophan (W41) in an aim to probe polarity/water environment near the edge of the heme-containing active site. 7-azatryptophan ((7-aza)Trp) is sensitive to environment polarity, while 2,7-azatryptophan ((2,7-aza)Trp) and 2,6-diazatryptophan ((2,6-aza)Trp) undergo excited-state water-catalyzed double and triple proton transfer, respectively, and are sensitive to the water network. The combination of their absorption, emission bands and the associated relaxation dynamics of these fluorescence probes, together with the Soret-band difference absorption and resonance Raman spectroscopy, lead us to unveil the water associated conformational mobility in the active site of APX. The results are suggestive of the existence of equilibrium between two different environments surrounding W41 in APX, i.e., the water-rich and water-scant forms with distinct fluorescence relaxation. Our results thus demonstrate for the first time the power of integrating multiple sensors (7-aza)Trp, (2,7-aza)Trp and (2,6-aza)Trp in probing the water environment of a specifically targeted Trp in proteins.

Homology Modeling and Molecular Dynamics Simulation Combined with X-ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases.

A combination of molecular dynamics (MD) simulations and X-ray scattering (SAXS) has emerged as the approach of choice for studying protein structures and dynamics in solution. This approach has potential applications for membrane proteins that neither are soluble nor form crystals easily. We explore the water-coupled dynamic structures of thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from scanning HPLC-SAXS measurements combined with MD ensemble analyses. Both proteins are heme-containing enzymes in the cytochrome P450 family, known as prostaglandin H2 (PGH2) isomerase, with counter-functions in regulation of platelet aggregation. Currently, the X-ray crystallographic structures of PGIS are available, but those for TXAS are not. The use of homology modeling of the TXAS structure with ns-µs explicit water solvation MD simulations allows much more accurate estimation of the configuration space with loop motion and origin of the protein behaviors in solution. In contrast to the stability of the conserved PGIS structure in solution, the pronounced TXAS flexibility has been revealed to have unstructured loop regions in connection with the characteristic P450 structural elements. The MD-derived and experimental-solution SAXS results are in excellent agreement. The significant protein internal motions, whole-molecule structures, and potential problems with protein folding, crystallization, and functionality are examined.

The In Situ Tryptophan Analogue Probes the Conformational Dynamics in Asparaginase Isozymes.

Dynamic water solvation is crucial to protein conformational reorganization and hence to protein structure and functionality. We report here the characterization of water dynamics on the L-asparaginase structural homology isozymes L-asparaginases I (AnsA) and II (AnsB), which are shown via fluorescence spectroscopy and dynamics in combination with molecular dynamics simulation to have distinct catalytic activity. By use of the tryptophan (Trp) analog probe 2,7-diaza-tryptophan ((2,7-aza)Trp), which exhibits unique water-catalyzed proton-transfer properties, AnsA and AnsB are shown to have drastically different local water environments surrounding the single Trp. In AnsA, (2,7-aza)Trp exhibits prominent green N(7)-H emission resulting from water-catalyzed excited-state proton transfer. In stark contrast, the N(7)-H emission is virtually absent in AnsB, which supports a water-accessible and a water-scant environment in the proximity of Trp for AnsA and AnsB, respectively. In addition, careful analysis of the emission spectra and corresponding relaxation dynamics, together with the results of molecular dynamics simulations, led us to propose two structural states associated with the rearrangement of the hydrogen-bond network in the vicinity of Trp for the two Ans. The water molecules revealed in the proximity of the Trp residue have semiquantitative correlation with the observed emission spectral variations of (2,7-aza)Trp between AnsA and AnsB. Titration of aspartate, a competitive inhibitor of Ans, revealed an increase in N(7)-H emission intensity in AnsA but no obvious spectral changes in AnsB. The changes in the emission profiles reflect the modulation of structural states by locally confined environment and trapped-water collective motions.

Probing the polarity and water environment at the protein-peptide binding interface using tryptophan analogues.

7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide Baa (LKWKKLLKLLKKLLKLG-NH2) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp-Baa and (2,7-aza)Trp-Baa, to study the calmodulin (CaM)-peptide interaction. Dramatic differences in the (7-aza)Trp-Baa and (2,7-aza)Trp-Baa fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. The affinity of the peptides for binding CaM followed the trend Baa (210±10 pM)<(7-aza)Trp-Baa (109±5 pM)<(2,7-aza)Trp-Baa (45±2 pM), showing moderate increase in binding affinity upon increasing the number of nitrogen atoms in the Trp analogue. The increased binding affinity may be due to the formation of more hydrogen bonds upon binding CaM for the Trp analogue with more nitrogen atoms. Importantly, the results demonstrate that (7-aza)Trp and (2,7-aza)Trp are excellent probes for exploring the environment at the interface of protein-peptide interactions.

Probing water environment of Trp59 in ribonuclease T1: insight of the structure-water network relationship.

In this study, we used the tryptophan analogue, (2,7-aza)Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)Trp. The resulting (2,7-aza)Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)Trp59, which catalyzes (2,7-aza)Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of α-helix and ß2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1.

Identification of kazinol Q, a natural product from Formosan plants, as an inhibitor of DNA methyltransferase.

DNA methylation plays a pivotal role in the epigenetic regulation of the transcription of a number of cancer-related genes, thereby representing an important target for cancer prevention and treatment. In our search for DNA methyltransferase (DNMT) inhibitors from Formosan plants, by screening against a library consisting of 12 structurally distinct natural products, we identified kazinol Q {4-[6-(1,1-dimethyl-allyl)-7-hydroxy-chroman-2-yl]-3,6-bis-(3-methyl-but-2-enyl)-benzene-1,2-diol} as an inhibitor of recombinant DNMT1 with IC50 of 7 µM. The effect of kazinol Q on DNMT inhibition was validated by its ability to reactivate the expression of a DNA methylation-silenced gene, E-cadherin, in MDA-MB-231 breast cancer cells. Moreover, kazinol Q suppressed the proliferation of MCF-7 breast and LNCaP prostate cancer cells, in part, through apoptosis induction. The role of DNMT1 inhibition in mediating kazinol Q's antiproliferative effect was supported by the protective effect of ectopic expression of DNMT1 on kazinol Q-induced cell death. Molecular modeling analysis suggests that kazinol Q inhibited DNMT activity by competing with cytosine binding, a mechanism similar to that described for (-)-epigallocatechin-3-gallate (EGCG). Relative to EGCG, kazinol Q exhibits several desirable features for drug development, including chemical stability and increased hydrophobicity, and might have therapeutic relevance to cancer treatment.

Probing water micro-solvation in proteins by water catalysed proton-transfer tautomerism.

Scientists have made tremendous efforts to gain understanding of the water molecules in proteins via indirect measurements such as molecular dynamic simulation and/or probing the polarity of the local environment. Here we present a tryptophan analogue that exhibits remarkable water catalysed proton-transfer properties. The resulting multiple emissions provide unique fingerprints that can be exploited for direct sensing of a site-specific water environment in a protein without disrupting its native structure. Replacing tryptophan with the newly developed tryptophan analogue we sense different water environments surrounding the five tryptophans in human thromboxane A2 synthase. This development may lead to future research to probe how water molecules affect the folding, structures and activities of proteins.

Vitamin E facilitates the inactivation of the kinase Akt by the phosphatase PHLPP1.

Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain-dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.