Interactions of the DNA Repair Enzyme Human Thymine DNA Glycosylase with Cognate and Noncognate DNA.

Glycosylases specifically recognize and flip their target base out of the DNA helix into the enzyme's active site. Our simulations show that a partially flipped state, already present in free DNA carrying a T:G mispair, becomes the more probable state compared to the closed state after binding of thymine DNA glycosylase (TDG). Paired thymine (T:A) or methyl-cytosine (mC:G) does not exhibit a partially flipped state in free or complexed DNA. Important enzyme-DNA interactions exhibit significant strength in the intrahelical and extrahelical TDG-DNA complexes. The computed binding free energy differences suggest these interactions account for the stabilization of the partially flipped state, thereby driving the T:G mispair toward base flip. In the fully flipped state, the cognate base thymine is significantly better accommodated in the enzyme's active site than noncognate bases are, suggesting the hydrolysis step as the last of several stages at which base recognition can be achieved.

Mechanism of the Glycosidic Bond Cleavage of Mismatched Thymine in Human Thymine DNA Glycosylase Revealed by Classical Molecular Dynamics and Quantum Mechanical/Molecular Mechanical Calculations.

Base excision of mismatched or damaged nucleotides catalyzed by glycosylase enzymes is the first step of the base excision repair system, a machinery preserving the integrity of DNA. Thymine DNA glycosylase recognizes and removes mismatched thymine by cleaving the C1'-N1 bond between the base and the sugar ring. Our quantum mechanical/molecular mechanical calculations of this reaction in human thymine DNA glycosylase reveal a requirement for a positive charge in the active site to facilitate C1'-N1 bond scission: protonation of His151 significantly lowers the free energy barrier for C1'-N1 bond dissociation compared to the situation with neutral His151. Shuttling a proton from His151 to the thymine base further reduces the activation free energy for glycosidic bond cleavage. Classical molecular dynamics simulations of the H151A mutant suggest that the mutation to the smaller, neutral, residue increases the water accessibility of the thymine base, rendering direct proton transfer from the bulk feasible. Quantum mechanical/molecular mechanical calculations of the glycosidic bond cleavage reaction in the H151A mutant show that the activation free energy is slightly lower than in the wild-type enzyme, explaining the experimentally observed higher reaction rates in this mutant.

Base flip in DNA studied by molecular dynamics simulationsof differently-oxidized forms of methyl-Cytosine.

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.

Characterization of a new class of androgen receptor antagonists with potential therapeutic application in advanced prostate cancer.

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts.

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.

Temperature dependence of the kinetic isotope effects in thymidylate synthase. A theoretical study.

In recent years, the temperature dependence of primary kinetic isotope effects (KIE) has been used as indicator for the physical nature of enzyme-catalyzed H-transfer reactions. An interactive study where experimental data and calculations examine the same chemical transformation is a critical means to interpret more properly temperature dependence of KIEs. Here, the rate-limiting step of the thymidylate synthase-catalyzed reaction has been studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) simulations in the theoretical framework of the ensemble-averaged variational transition-state theory with multidimensional tunneling (EA-VTST/MT) combined with Grote-Hynes theory. The KIEs were calculated across the same temperature range examined experimentally, revealing a temperature independent behavior, in agreement with experimental findings. The calculations show that the H-transfer proceeds with ∼91% by tunneling in the case of protium and ∼80% when the transferred protium is replaced by tritium. Dynamic recrossing coefficients are almost invariant with temperature and in all cases far from unity, showing significant coupling between protein motions and the reaction coordinate. In particular, the relative movement of a conserved arginine (Arg166 in Escherichia coli ) promotes the departure of a conserved cysteine (Cys146 in E. coli ) from the dUMP by polarizing the thioether bond thus facilitating this bond breaking that takes place concomitantly with the hydride transfer. These promoting vibrations of the enzyme, which represent some of the dimensions of the real reaction coordinate, would limit the search through configurational space to efficiently find those decreasing both barrier height and width, thereby enhancing the probability of H-transfer by either tunneling (through barrier) or classical (over-the-barrier) mechanisms. In other words, the thermal fluctuations that are coupled to the reaction coordinate, together with transition-state geometries and tunneling, are the same in different bath temperatures (within the limited experimental range examined). All these terms contribute to the observed temperature independent KIEs in thymidylate synthase.

Application of Grote-Hynes theory to the reaction catalyzed by thymidylate synthase.

A theoretical study of dynamic effects on the rate-limiting step of the thymidylate synthase catalyzed reaction has been carried out by means of Grote-Hynes theory, successfully predicting the values of the recrossing effects for a chemical reaction that involves the transfer of a classical light particle. The transmission coefficients, obtained at 278, 293, 303, and 313 K, are almost invariant and in all cases far from unity, revealing a significant coupling of the environment motions and the reaction coordinate. Nevertheless, their energetic contribution to the activation free energy represents less than 0.50 kcal/mol for each of the four tested temperatures. Calculation of the transmission coefficient for the isotopically labeled hydride transfer has rendered almost the same values, in agreement with the experimentally observed temperature-independent KIEs. Fourier transform of the time-dependent friction kernel at these four temperatures has allowed obtaining the transition-state friction spectra, which present very small dependence with temperature. Their analysis has led to the identification of some key vibrational modes governing the coupling between the reaction coordinate and the protein environment, thus identifying the relevant motions in the active site and obtaining a full picture of the role of each amino acid.

Theoretical study of the temperature dependence of dynamic effects in thymidylate synthase.

A theoretical study of the temperature dependence of dynamic effects in the rate limiting step of the reaction catalyzed by thymidylate synthase is presented in this paper. From hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations of transition state structures within a fully flexible molecular model, free downhill molecular dynamics trajectories have been performed at four different temperatures. The analysis of the reactive and non-reactive trajectories in the enzyme environment has allowed us to study the geometric and electronic coupling between the substrate, the cofactor and the protein. The results show how the contribution of dynamic effects to the rate enhancement measured by the transmission coefficients is, at the four studied temperatures, negligible. Nevertheless, the rare event trajectories performed have shown how the hydride transfer and the scission of the conserved active site cysteine residue (Cys146 in E. coli) take place in a concerted but asynchronous way; the latter takes place once the transfer has occurred. The analysis of the dynamics of the protein reveals also how the relative movements of some amino acids, especially Arg166, and a water molecule, promotes the departure of the Cys146 from the dUMP. Finally, it seems that the protein environment creates an almost invariant electric field in the active site of the protein that stabilizes the transition state of the reaction, thus reducing the free energy barrier.

QM/MM study of thymidylate synthase: enzymatic motions and the temperature dependence of the rate limiting step.

Thymidylate synthase (TS) is an enzyme that catalyzes a complex cascade of reactions. A theoretical study of the reduction of an exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H(4)folate), to form 2'-deoxyuridine 5'-monophosphate (dTMP) and 7,8-dihydrofolate (H(2)folate), has been carried out using hybrid quantum mechanics/molecular mechanics methods. This step is of special interest because it is the rate-limiting step of the reaction catalyzed by TS. The acceptor of this hydride is an intermediate that is covalently bound to the enzyme via a thioether bond to an overall conserved active site cysteine residue (Cys146 in Escherichia coli). Heretofore, whether the hydride transfer precedes the thiol abstraction that releases the product from the enzyme or whether these two processes are concerted has been an open question. We have examined this step in terms of free energy surfaces obtained at the same temperatures we previously used in experimental studies of this mechanistic step (273-313 K). Analysis of the results reveals that substantial features of the reaction and the nature of the H-transfer seem to be temperature independent, in agreement with our experimental data. The findings also indicate that the hydride transfer and the scission of Cys146 take place in a concerted but asynchronous fashion. This 1,3-S(N)2 substitution is assisted by arginine 166 and several other arginine residues in the active site that polarize the carbon-sulfur bond and stabilize the charge transferred from cofactor to substrate. Finally, the simulation elucidates the molecular details of the enzyme's motion that brings the system to its transition state and, in accordance with the experimental data, indicates that this "tunneling ready" conformation is temperature independent.

QM/MM simulations for methyl transfer in solution and catalysed by COMT: ensemble-averaging of kinetic isotope effects.

Sampling of structures from QM/MM molecular dynamics reveals distinct families of reactant-state conformers and yields kinetic isotope effects for reactions in enzyme active sites and in solution, averaged over thermal fluctuations of the environment, that allows meaningful comparison of computed with experimental values.

A quantum mechanics/molecular mechanics study of the protein-ligand interaction of two potent inhibitors of human O-GlcNAcase: PUGNAc and NAG-thiazoline.

O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.

A quantum mechanics/molecular mechanics study of the catalytic mechanism of the thymidylate synthase.

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.