A modified HSP70 inhibitor shows broad activity as an anticancer agent.

The stress-induced HSP70 is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in nontransformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here, we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70 and requires the C-terminal helical "lid" of this protein (amino acids 573-616) to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure-activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B-cell lymphoma, compared with the parent compound (P = 0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the anaphase promoting complex/cyclosome (APC/C) in cell-free extracts, and induce G2-M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anticancer compound with several notable mechanisms of action.

QM/MM X-ray refinement of zinc metalloenzymes.

Zinc metalloenzymes play an important role in biology. However, due to the limitation of molecular force field energy restraints used in X-ray refinement at medium or low resolutions, the precise geometry of the zinc coordination environment can be difficult to distinguish from ambiguous electron density maps. Due to the difficulties involved in defining accurate force fields for metal ions, the QM/MM (quantum-mechanical/molecular-mechanical) method provides an attractive and more general alternative for the study and refinement of metalloprotein active sites. Herein we present three examples that indicate that QM/MM based refinement yields a superior description of the crystal structure based on R and R(free) values and on the inspection of the zinc coordination environment. It is concluded that QM/MM refinement is an useful general tool for the improvement of the metal coordination sphere in metalloenzyme active sites.

A Mixed QM/MM Scoring Function to Predict Protein-Ligand Binding Affinity.

Computational methods for predicting protein-ligand binding free energy continue to be popular as a potential cost-cutting method in the drug discovery process. However, accurate predictions are often difficult to make as estimates must be made for certain electronic and entropic terms in conventional force field based scoring functions. Mixed quantum mechanics/molecular mechanics (QM/MM) methods allow electronic effects for a small region of the protein to be calculated, treating the remaining atoms as a fixed charge background for the active site. Such a semi-empirical QM/MM scoring function has been implemented in AMBER using DivCon and tested on a set of 23 metalloprotein-ligand complexes, where QM/MM methods provide a particular advantage in the modeling of the metal ion. The binding affinity of this set of proteins can be calculated with an R(2) of 0.64 and a standard deviation of 1.88 kcal/mol without fitting and 0.71 and a standard deviation of 1.69 kcal/mol with fitted weighting of the individual scoring terms. In this study we explore using various methods to calculate terms in the binding free energy equation, including entropy estimates and minimization standards. From these studies we found that using the rotational bond estimate to ligand entropy results in a reasonable R(2) of 0.63 without fitting. We also found that using the ESCF energy of the proteins without minimization resulted in an R(2) of 0.57, when using the rotatable bond entropy estimate.

A Combined QM/MM Poisson-Boltzmann Approach.

A method of solving the mixed quantum mechanical/molecular mechanical (QM/MM) Hamiltonian in solution, using the Poisson-Boltzmann (PB) equation to calculate partial charges and solvation free energies, is presented. This method combines a linear scaling divide and conquer semiempirical algorithm with the PB equation in a QM/MM framework, allowing only a specified region's charges to be polarized by the solvent while using fixed charges from a MM force field for the remaining system. This can save time over a full QM implementation, only requiring self-consistency to be achieved in a small QM region, while giving comparable results. The solvation free energy of pentapeptides capped with an acetyl group (ACE) at the N-terminus and an N-methylamine group at the C-terminus (NME) was used to study the accuracy of this method as well as several small protein systems. The solvation free energies for the QM/MM implementation compare well with a full QM treatment of the same system, giving reasonable representations for the solvation free energy of the entire system independent of the QM region's size. In the case of the pentapeptides, the average error was only 4.9 kcal/mol with the smallest QM region. This mixed method will allow an accurate description of solvation effects in an area of interest, such as an active site, using mixed QM/MM Hamiltonians. Possible applications for this method include protein-ligand binding and reaction mechanism studies.

Critical assessment of quantum mechanics based energy restraints in protein crystal structure refinement.

A critical evaluation of the performance of X-ray refinement protocols using various energy functions is presented using the bovine pancreatic trypsin inhibitor (BPTI) protein. The four potential energy functions we explored include: (1) fully quantum mechanical calculations; (2) one based on an incomplete molecular mechanics (MM) energy function employed in the Crystallography and NMR System (CNS) with empirical parameters developed by Engh and Huber (EH), which lacks electrostatic and attractive van der Waals terms; (3) one based on a complete MM energy function (AMBER ff99 parameter set); and (4) the same as 3, with the addition of a Generalized Born (GB) implicit solvation term. The R, R (free), real space R values of the refined structures and deviations from the original experimental structure were used to assess the relative performance. It was found that at 1 Angstrom resolution the physically based energy functions 1, 3, and 4 performed better than energy function 2, which we attribute to the better representation of key interactions, particularly electrostatics. The observed departures from the experimental structure were similar for the refinements with physically based energy functions and were smaller than the structure refined with EH. A test refinement was also performed with the reflections truncated at a high-resolution cutoff of 2.5 Angstrom and with random perturbations introduced into the initial coordinates, which showed that low-resolution refinements with physically based energy functions held the structure closer to the experimental structure solved at 1 Angstrom resolution than the EH-based refinements.

Assigning the protonation states of the key aspartates in ß-Secretase using QM/MM X-ray structure refinement.

ß-Secretase, a.k.a. ß-APP cleaving enzyme (BACE), is an aspartyl protease that has been implicated as a key target in the pathogenesis of Alzheimer's disease (AD). The identification of the protonation states of the key aspartates in ß-secretase is of great interest both in understanding the reaction mechanism and in guiding the design of drugs against AD. However, the resolutions of currently available crystal structures for BACE are not sufficient to determine the hydrogen atom locations. We have assigned the protonation states of the key aspartates using a novel method, QM/MM X-ray refinement. In our approach, an energy function is introduced to the refinement where the atoms in the active site are modeled by quantum mechanics (QM) and the other atoms are represented by molecular mechanics (MM). The gradients derived from the QM/MM energy function are combined with those from the X-ray target to refine the crystal structure of a complex containing BACE and an inhibitor. A total number of 8 protonation configurations of the aspartyl dyad were considered and QM/MM X-ray refinements were performed for all of them. The relative stability of the refined structures was scored by constructing the thermodynamic cycle using the energetics calculated by fully quantum mechanical self-consistent reaction field (QM/SCRF) calculations. While all 8 refined structures fit the observed electron density about equally well, we find the mono-protonated configurations to be strongly favored energetically, especially the configuration with the inner oxygen of Asp32 protonated and the hydroxyl of the inhibitor pointing towards Asp228. It was also found that these results depend on the constraints imposed by the X-ray data. We suggest that one of the strengths of this approach is that the resulting structures are a consensus of theoretical and experimental data and remark on the significance of our results in structure based drug design and mechanistic studies.