Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT 90 ) and time to 90% Ca 2+ transient decay (DT 90 ), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT 90 and partially normalized DT 90 , suggesting improved Ca 2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca 2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

Expression and significance of aquaporin-4 in thyroid carcinoma.

OBJECTIVE: To investigate the expression of aquaporin-4 (AQP4) in thyroid carcinoma (TC) and explore its clinical significance. MATERIALS AND METHODS: The formalin-fixed paraffin-embedded specimens including 275 TC cancer tissues, 258 corresponding paracancerous thyroid tissues and their clinicopathologic data were retrospectively analyzed. Immunohistochemical EnVision two-step method was used to detect the expression of AQP4 in the cancer tissues and adjacent thyroid tissues, and its clinical significance was analyzed. RESULTS: AQP4 could be expressed in both TC cancer tissues and paracancerous thyroid tissues. In TC cancer tissues, the positive expression rate was 99.3% (273/275), and the positive expression rate was 86.4% (223/258) in paracancerous thyroid tissues. The expression level of AQP4 in cancer tissues was significantly higher than that in paracancerous thyroid tissues, and the difference was statistically significant (P < 0.05). The positive expression rates of AQP4 in papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC) and undifferentiated thyroid carcinoma (UTC) were 99.2% (258/260), 100.0% (6/6), 100.0% (6/6) and 100.0% (3/3), respectively and there was little difference in different types of TC. Analysis of relationship between expression level of AQP4 in 275 TC cancer tissues and 260 PTC cancer tissues and clinicopathologic characteristics of patients was not significant correlation (P > 0.05). Among the 275 patients, one (0.4%, 1/275) was diagnosed as neuromyelitis optica spectrum disorder (NMOSD) associated with TC. CONCLUSIONS: AQP4 is generally expressed in TC cancer tissues and paracancerous thyroid tissues. Expression level of AQP4 in cancer tissues was significantly higher than that in paracancerous thyroid tissue. Expression level of AQP4 in TC cancer tissues is not related to the clinicopathological characteristics of the patients. Paraneoplastic NMOSD caused by TC is rare, and whether its specific pathogenesis is related to the expression of AQP4 in TC still needs further study.

Long-term and inter-monthly dynamics of aquatic vegetation and its relation with environmental factors in Taihu Lake, China.

This paper discussed the long-term and inter-monthly variation in the distribution area of aquatic macrophytes in Taihu Lake, as well as the relationship between these variations and environmental factors. The findings were of great significance to the protection and environmental remediation of lake ecosystems. This paper presented data from 92 periods during 1980 to 2017 on the distribution area of aquatic macrophytes (including submerged macrophytes and floating-leaved macrophytes, but excluding emergent macrophytes) in Taihu Lake. Data were acquired by remote-sensing and subsequent image interpretation. The analysis of the inter-monthly variation indicated that the area occupied by aquatic macrophytes first increased and then decreased from January to December. Specifically, the distribution area was very small from January to March, began to increase gradually from April to August, reached its maximum in September, and decreased gradually from October to December. The analysis of the long-term variation showed that the distribution and area of aquatic macrophytes experienced two stages during the years 1980 to 2017: 1) gradual increase, 2) sharp decrease. In the first stage (1980 to 2014), the area occupied by aquatic macrophytes increased by 9 times, the maximum distribution area was 206.27 km2 (in May), 307.92 km2 (in September) and 277.33 km2 (in October). In the second stage (2015 to 2017), the distribution area of aquatic macrophytes decreased sharply to 50 km2 or less. The distribution area of aquatic macrophytes during the months of January to December had a significant positive correlation with monthly average temperature, CODMn value, secchi disk depth(SDD), area of cyanobacteria and chlorophyll-a (Chl-a) concentration, a significant negative correlation with water quality indices such as dissolved oxygen (DO) value and NH3-N concentration, but no significant correlation with water quality indices such as pH values and total suspended matter (TSM) concentration. The distribution area of aquatic macrophytes from 1980 to 2017 had a significant positive correlation with annual average temperature, annual minimum water level, pH value, SDD, area of cyanobacteria and Chl-a concentration, but no significant correlation with water quality indices such as DO value, CODMn value, NH3-N concentration and TSM concentration. The sharp decrease in the distribution area of aquatic macrophytes in 2015 and subsequent years was primarily due to the mechanized salvage of aquatic plants.

Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation.

cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac-specific N-terminus (cTnI(R21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions. Here, we tested whether this also occurs for cTnI(P83S). cTnI(P83S) increased both Ca(2+) binding affinity to cTn (KCa) and affinity of cTnC for cTnI (KC-I), and eliminated the reduction of KCa and KC-I observed for phosphorylated-cTnI(WT). In isolated myofibrils, cTnI(P83S) maintained maximal tension (TMAX) and Ca(2+) sensitivity of tension (pCa50). For cTnI(WT) myofibrils, PKA-mediated phosphorylation decreased pCa50 and sped up the slow-phase relaxation (especially for those Ca(2+) conditions that heart performs in vivo). Those effects were blunted for cTnI(P83S) myofibrils. Molecular-dynamics simulations suggested cTnI(P83S) moderately inhibited an intrasubunit interaction formation between inhibitory-peptide and N-terminus, but this "blunting" effect was weaker than that with cTnI(R146G) or cTnI(R21C). In summary, cTnI(P83S) has similar effects as other HCM-associated cTnI mutations on troponin and myofibril function even though it is in the I-T arm of cTnI.

[Clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumor].

OBJECTIVE: To investigate clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GIST). METHODS: Immunohistochemical EnVision technique was used to assess the expression of succinate dehydrogenase subunit B (SDHB) in 192 cases of GIST. Cases of SDH-deficient GIST were further evaluated for the presence of CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations with clinical followed-up data. RESULTS: Seven of the 192 cases showed SDHB-deficiency (3.6%, 7/192). The patients ranged in age from 35 to 84 years (median=56 years; mean=60 years). Four were male and three were female. Six tumors involved stomach and one involved mesentery. Histopathologic features of SDHB-deficient GIST included four cases of mixed-cell type and three of epithelioid cell type. The tumors commonly involved muscularis propria of the stomach as multiple nodules, creating a plexiform pattern. The tumors had high cellularity with cytoplasmic vacuolization. Five cases developed lymph node metastases including one also metastasizing to liver and pancreas. Two cases showed no evidence of metastasis. None of the 7 cases of the SDHB-deficient GIST had CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations. Three of the seven SDHB-deficient GIST cases had followed-up data: two did not recur and one died after 24 months of surgery of unknown cause. CONCLUSION: SDHB-deficient GIST has characteristic clinicopathologic features with wide-type CKIT gene and a favorable prognosis.

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility.

Cardiac troponin (cTn) acts as a pivotal regulator of muscle contraction and relaxation and is composed of three distinct subunits (cTnC: a highly conserved Ca(2+) binding subunit, cTnI: an actomyosin ATPase inhibitory subunit, and cTnT: a tropomyosin binding subunit). In this mini-review, we briefly summarize the structure-function relationship of cTn and its subunits, its modulation by PKA-mediated phosphorylation of cTnI, and what is known about how these properties are altered by hypertrophic cardiomyopathy (HCM) associated mutations of cTnI. This includes recent work using computational modeling approaches to understand the atomic-based structural level basis of disease-associated mutations. We propose a viewpoint that it is alteration of cTnC-cTnI interaction (rather than the Ca(2+) binding properties of cTn) per se that disrupt the ability of PKA-mediated phosphorylation at cTnI Ser-23/24 to alter contraction and relaxation in at least some HCM-associated mutations. The combination of state of the art biophysical approaches can provide new insight on the structure-function mechanisms of contractile dysfunction resulting cTnI mutations and exciting new avenues for the diagnosis, prevention, and even treatment of heart diseases.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.

Contractile properties of developing human fetal cardiac muscle.

KEY POINTS: The contractile properties of human fetal cardiac muscle have not been previously studied. Small-scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well-controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59-134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell-derived cardiomyocytes. ABSTRACT: Little is known about the contractile properties of human fetal cardiac muscle during development. Understanding these contractile properties, and how they change throughout development, can provide valuable insight into human heart development, and provide a framework to study the early stages of cardiac diseases that develop in utero. We characterized the contractile properties of isolated human fetal cardiac myofibrils across 8-19 weeks of gestation. Mechanical measurements revealed that in early stages of gestation there is low specific force and slow rates of force development and relaxation, with increases in force and the rates of activation and relaxation as gestation progresses. The duration and slope of the initial, slow phase of relaxation, related to myosin detachment and thin filament deactivation rates, decreased with gestation age. F-actin sliding on human fetal cardiac myosin-coated surfaces slowed significantly from 108 to 130 days of gestation. Electron micrographs showed human fetal muscle myofibrils elongate and widen with age, but features such as the M-line and Z-band are apparent even as early as day 52. Protein isoform analysis revealed that ß-myosin is predominantly expressed even at the earliest time point studied, but there is a progressive increase in expression of cardiac troponin I (TnI), with a concurrent decrease in slow skeletal TnI. Together, our results suggest that cardiac myofibril force production and kinetics of activation and relaxation change significantly with gestation age and are influenced by the structural maturation of the sarcomere and changes in contractile filament protein isoforms.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation.

Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during ß-adrenergic stimulation. Here, we tested how cTnI(R146G) or cTnI(R21C) influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca(2+) binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnI(WT). cTnI(WT), cTnI(R146G), and cTnI(R21C) were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca(2+) sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca(2+) levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI(R146G) and cTnI(R21C) blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.

Effects of HCM cTnI mutation R145G on troponin structure and modulation by PKA phosphorylation elucidated by molecular dynamics simulations.

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI(1-39)), is a target for phosphorylation by protein kinase A (PKA) during ß-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl(137-147)) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca(2+) handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), cTnI-R145G/S23D/S24D Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), and cTnI-R145G/PS23/PS24 Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca(2+)-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca(2+) coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during ß-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during ß-adrenergic stimulation.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure.

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.

Computational studies of the effect of the S23D/S24D troponin I mutation on cardiac troponin structural dynamics.

During ß-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1-40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.

PKA phosphorylation of cardiac troponin I modulates activation and relaxation kinetics of ventricular myofibrils.

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for ß-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca(2+) levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca(2+) concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca(2+) activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca(2+), and reduced the Ca(2+) sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca(2+)-cTn binding affinity and C-I interaction were monitored. Ca(2+) binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca(2+). These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during ß-adrenergic stimulation.

A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells.

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.

Unraveling the allosteric inhibition mechanism of PTP1B by free energy calculation based on umbrella sampling.

Protein tyrosine phosphatase 1B (PTP1B) is a promising target for the treatment of obesity and type II diabetes. Allosteric inhibitors can stabilize an active conformation of PTP1B by hindering the conformational transition of the WPD loop of PTP1B from the open to the closed state. Here, the umbrella sampling molecular dynamics (MD) simulations were employed to compute the reaction path of the conformational transition of PTP1B, and the snapshots extracted from the MD trajectory were clustered into 58 conformational groups based on the key conformational parameter. Then, the impact of the conformational change of the WPD loop on the interactions between the allosteric site of PTP1B and an allosteric inhibitor BB3 was explored by using the MM/GBSA binding free energy calculations and free energy decomposition analysis. The simulation results show that the binding free energy of BB3 increases gradually from the open to the closed conformation of the WPD loop, providing the molecular mechanism of allosteric inhibition. Correlation analysis of the different energy terms indicates that the allosteric inhibitor with more negative van der Waals contribution cannot only exhibit stronger binding affinity but also hinder the swing of the WPD loop more effectively. Besides, it is found that the energy contribution of Lys292 in the α7 helix undergoes significant change, which reveals that Lys292 is not only the key residue for ligand binding but also plays an important role in hindering the conformational change of the WPD loop.

Structural basis of specific binding between Aurora A and TPX2 by molecular dynamics simulations.

In the present study, the impacts of G198N and W128F mutations on the recognition between Aurora A and targeting protein of Xenopus kinesin-like protein 2 (TPX2) were investigated using molecular dynamics (MD) simulations, free energy calculations, and free energy decomposition analysis. The predicted binding free energy of the wild-type complex is more favorable than those of three mutants, indicating that both single and double mutations are unfavorable for the Aurora A and TPX2 binding. It is also observed that the mutations alternate the binding pattern between Aurora A and TPX2, especially the downstream of TPX2. An intramolecular hydrogen bond between the atom OD of Asp11(TPX2) and the atom HE1 of Trp34(TPX2) disappear in three mutants and thus lead to the instability of the secondary structure of TPX2. The combination of different molecular modeling techniques is an efficient way to understand how mutation has impacts on the protein-protein binding and our work gives valuable information for the future design of specific peptide inhibitors for Aurora A.

The molecular mechanism studies of chirality effect of PHA-739358 on Aurora kinase A by molecular dynamics simulation and free energy calculations.

Aurora kinase family is one of the emerging targets in oncology drug discovery and several small molecules targeting aurora kinases have been discovered and evaluated under early phase I/II trials. Among them, PHA-739358 (compound 1r) is a 3-aminopyrazole derivative with strong activity against Aurora A under early phase II trial. Inhibitory potency of compound 1r (the benzylic substituent at the pro-R position) is 30 times over that of compound 1s (the benzylic substituent at the pro-S position). In present study, the mechanism of how different configurations influence the binding affinity was investigated using molecular dynamics (MD) simulations, free energy calculations and free energy decomposition analysis. The predicted binding free energies of these two complexes are consistent with the experimental data. The analysis of the individual energy terms indicates that although the van der Waals contribution is important for distinguishing the binding affinities of these two inhibitors, the electrostatic contribution plays a more crucial role in that. Moreover, it is observed that different configurations of the benzylic substituent could form different binding patterns with protein, thus leading to variant inhibitory potency of compounds 1r and 1s. The combination of different molecular modeling techniques is an efficient way to interpret the chirality effects of inhibitors and our work gives valuable information for the chiral drug design in the near future.

Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations.

Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.

Octathienyl/phenyl-substituted zinc phthalocyanines J-aggregated through conformational planarization.

A novel family of thiophene-decorated phthalocyanines (M-TPcs) was efficiently synthesized, during which the key intermediate of these compounds was purified through a chemical approach. In the optimized geometries of M-TPcs, the peripherally linked thiophene rings are tilted from the Pc core, which oppose aggregation considering the mutual steric hindrance. However, Zn-TPc formed J-aggregates in many solvents while Ni-TPc and Cu-TPc did not. Octaphenyl-substituted zinc Pc (Zn-PPc) showed similar J-aggregation behavior as Zn-TPc. This unusual J-aggregation was attributed to the conformational planarization of the corresponding molecules.