Stephen Quirk

Matrix metalloproteinase inhibitors

SummaryThe matrix metalloproteinases (MMPs) are a family of closely related, zinc-dependent proteolytic enzymes. Collectively, they are capable of degrading all the components of the extracellular matrix and as such are involved in a number of physiological and pathological processes.The extracellular matrix is the principal barrier to tumour growth and spread, and there is evidence that MMPs play a role in the processes of tumour growth and metastasis. Therefore, inhibitors of MMPs may be of value in the treatment of malignant disease. There exist naturally occurring inhibitors of these enzymes known as ‘tissue inhibitors of MMPs’, or TIMPs. Although there have been considerable preclinical studies on these inhibitors, they are as yet unavailable for use as therapeutic drugs. Research in this field has focused largely on the development of low molecular weight (<500D) synthetic inhibitors of MMPs.In this review we focus on the various subgroups of MMP inhibitors now available, their preclinical evaluation and the limited information available from preliminary clinical trials. We comment on the suitability of the preclinical models used and the difficulty in designing clinical trials of these drugs. We focus on future developments which may involve the use of these drugs in combination with existing chemotherapeutic regimens to achieve a synergistic effect.

Adaptive Steered Molecular Dynamics of the Long-Distance Unfolding of Neuropeptide Y.

Neuropeptide Y (NPY) has been found to adopt two stable conformations in vivo: (1) a monomeric form called the PP-fold in which a polyproline tail is folded onto an α-helix via a β-turn and (2) a dimeric form of the unfolded proteins in which the α-helices interact with each other via side chains. The transition pathway and rates between the two conformations remain unknown and are important to the nature of the binding of the protein. Toward addressing this question, the present work suggests that the unfolding of the PP-fold is too slow to play a role in NPY monomeric binding unless the receptor catalyzes it to do so. Specifically, the dynamics and structural changes of the unfolding of a monomeric NPY protein have been investigated in this work. Temperature accelerated molecular dynamics (MD) simulations at 500 K under constant (N,V,E) conditions suggests a hinge-like unraveling of the tail rather than a random unfolding. The free energetics of the proposed unfolding pathway have been described using an adaptive steered MD (SMD) approach at various temperatures. This approach generalizes the use of Jarzynskis equality through a series of stages that allows for better convergence along nonlinear and long-distance pathways. Results acquired using this approach provide a potential of mean force (PMF) with narrower error bars and are consistent with some of the earlier reports on the qualitative behavior of NPY binding.

Adaptive steered molecular dynamics: Validation of the selection criterion and benchmarking energetics in vacuum

The potential of mean force (PMF) for stretching decaalanine in vacuum was determined earlier by Park and Schulten [J. Chem. Phys. 120, 5946 (2004)] in a landmark article demonstrating the efficacy of combining steered molecular dynamics and Jarzynskis nonequilibrium relation. In this study, the recently developed adaptive steered molecular dynamics (ASMD) algorithm [G. Ozer, E. Valeev, S. Quirk, and R. Hernandez, J. Chem. Theory Comput. 6, 3026 (2010)] is used to reproduce the PMF of the unraveling of decaalanine in vacuum by averaging over fewer nonequilibrium trajectories. The efficiency and accuracy of the method are demonstrated through the agreement with the earlier work by Park and Schulten, a series of convergence checks compared to alternate SMD pulling strategies, and an analytical proof. The nonequilibrium trajectories obtained through ASMD have also been used to analyze the intrapeptide hydrogen bonds along the stretching coordinate. As the decaalanine helix is stretched, the initially stabilized i → i + 4 contacts (α-helix) is replaced by i → i + 3 contacts (3(10)-helix). No significant formation of i → i + 5 hydrogen bonds (π-helix) is observed.

Thermodynamics of Decaalanine Stretching in Water Obtained by Adaptive Steered Molecular Dynamics Simulations

The nonequilibrium stretching of decaalanine in vacuum using steered molecular dynamics and Jarzynskis relation led to the landmark determination of its potential of mean force by Park and Schulten (Chem. Phys. 2004). In so doing, the relative thermodynamics of the hydrogen-bond contacts and the entropy of the chain were quantified through the reversible work, the potential of mean force (PMF). A recently developed adaptive steered molecular dynamics algorithm (Ozer et al. J. Chem. Theory Comput. 2010) has now made it possible to determine the thermodynamics, PMF, of the stretching of decaalanine in a model solvent of TIP3P water molecules. The loss of internal hydrogen bonds and the formation of hydrogen bonds between the peptide and the solvent has also been tracked with the corresponding stabilization in the PMF. As in the vacuum, most of the thermodynamic penalty to unravel the chain in solvent occurs during the regime when the internal hydrogen bonds are broken. The formation of hydrogen bonds with the solvent provides a significant stabilization not seen in vacuum, reducing the total energy cost to unravel by nearly a factor of 2.

Triggered release of small molecules from proteinoid microspheres

Proteinoid microspheres (PM) are formed by the thermal condensation of amino acids. They have been useful to further evolutionary theory, as catalysts for some biochemical reactions, but they have not been overly useful as controlled delivery agents. It is possible however to construct PMs that contain organic small molecules in the interior space. This means that a PM could be used as a delivery agent, if a suitable method could be discovered to cause the release of the internal material. This report describes the formation of a PM that includes a molecular bridging agent that can be removed in a reducing environment. Removal of the bridge opens a hole or window in the PM that allows the interior material to escape. The rate at which the interior material is released from the PM can be controlled by the size of the window or by the reduction potential in the environment. These PMs can be used to temporally treat a variety of complications including wounds (chronic or acute) by delivering a sequestered reagent in a controlled manner and are advantageous in that amino acids are the primary delivery vehicle breakdown product.

The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug-Polymer Conjugates.

Degradation reactions on diclofenac-monoglycerides (3a,b), diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c), diclofenac (1), and diclofenac lactam (4) were performed at 37 °C in isotonic buffer solutions (apparent pH range 1-8) containing varying concentrations of acetonitrile (ACN). The concentration remaining of each analyte was measured versus time. Diclofenac-monoglycerides and diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) were both found to undergo facile and complete hydrolysis in pH 7.4 isotonic phosphate buffer/10% ACN. Under mildly acidic, neutral or alkaline conditions, diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) had the fastest hydrolysis rate (t1/2 = 3.23 h at pH 7.4), with simultaneous formation of diclofenac lactam (4) and diclofenac (1). Diclofenac-monoglycerides (3a,b) hydrolyzed more slowly under the same conditions, to again yield both diclofenac (1) and diclofenac lactam (4). There was also transesterification of diclofenac-2-monoglyceride (3b) to its regioisomer, diclofenac-1-monoglyceride (3a) across the pH range. Diclofenac was shown to be stable in neutral or alkaline conditions but cyclized to form the lactam (4) in acidic conditions. Conversely, the lactam (4) was stable under acidic conditions but was converted to an unknown species under alkaline or neutral conditions.

Constrained Unfolding of a Helical Peptide: Implicit versus Explicit Solvents

Steered Molecular Dynamics (SMD) has been seen to provide the potential of mean force (PMF) along a peptide unfolding pathway effectively but at significant computational cost, particularly in all-atom solvents. Adaptive steered molecular dynamics (ASMD) has been seen to provide a significant computational advantage by limiting the spread of the trajectories in a staged approach. The contraction of the trajectories at the end of each stage can be performed by taking a structure whose nonequilibrium work is closest to the Jarzynski average (in naive ASMD) or by relaxing the trajectories under a no-work condition (in full-relaxation ASMD—namely, FR-ASMD). Both approaches have been used to determine the energetics and hydrogen-bonding structure along the pathway for unfolding of a benchmark peptide initially constrained as an α-helix in a water environment. The energetics are quite different to those in vacuum, but are found to be similar between implicit and explicit solvents. Surprisingly, the hydrogen-bonding pathways are also similar in the implicit and explicit solvents despite the fact that the solvent contact plays an important role in opening the helix.

Determining the Energetics of Small β-Sheet Peptides using Adaptive Steered Molecular Dynamics.

Mechanically driven unfolding is a useful computational tool for extracting the energetics and stretching pathway of peptides. In this work, two representative β-hairpin peptides, chignolin (PDB: 1UAO ) and trpzip1 (PDB: 1LE0 ), were investigated using an adaptive variant of the original steered molecular dynamics method called adaptive steered molecular dynamics (ASMD). The ASMD method makes it possible to perform energetic calculations on increasingly complex biological systems. Although the two peptides are similar in length and have similar secondary structures, their unfolding energetics are quite different. The hydrogen bonding profile and specific residue pair interaction energies provide insight into the differing stabilities of these peptides and reveal which of the pairs provides the most significant stabilization.

Triggered release from peptide-proteinoid microspheres.

Proteinoid microspheres (PM) are unusual polymers formed by the thermal condensation of amino acids. Although they have been studied for over 60 years, they are only now beginning to garner interest as controlled release agents. Although they are very biocompatible, it has been problematic to design useful triggers that release small molecules from PM interiors. This has severely limited their usefulness. In the present study, short peptides have been successfully incorporated into PMs during their formation. The resulting hybrid peptide-PMs can release their interior content when hydrolyzed by a proteinase. Specifically, if a matrix metalloproteinase (MMP) cleavage site peptide is incorporated into a PM, the peptide-PM will release interior contents only in the presence of the MMP recognizing the cleavage peptide. The release rate can be determined by the concentration of the peptide in the PM synthesis mixture. This potentially makes peptide-PMs useful for delivering inhibitors or drugs into acute and chronic wounds, periodontal disease sites, and other disease states involving the fine-tuned regulation of proteinases.

Optically tracked, single-coil, scanning magnetic induction tomography

Recent work has shown the feasibility of single-coil, magnetic induction tomography, for visualizing a 3D distribution of electrical conductivity in portions of the human body. Loss is measured in a single, planar coil consisting of concentric circular loops while the coil is relocated to various non-redundant positions and orientations in the vicinity of the target. These loss values, together with measured coil position and orientation, are processed by a quantitative mapping equation that enables reconstruction of an electrical conductivity image. Up until now, the position of the coil had to be established by a template, which required assignment of locations for the coil to visit without necessarily giving any prior consideration to target geometry. We have now added optical tracking to our existing single-coil device so that position and orientation are tracked automatically, allowing collection of coil loss data at arbitrary positions or orientations as needed. Optical tracking is accomplished via a set of IR reflective spheres mounted on the same enclosure that supports the coil. Position for a select sphere within the set, together with the four quaternions specifying optical body orientation, is fed to a laptop at the same time coil loss data is streamed to the same laptop via Bluetooth. The coil center can be tracked with sub-millimeter accuracy while orientation angle is known to a fraction of a degree. This work illustrates the use of single-coil MIT in full, position-orientation-tracked scan mode while imaging laboratory phantoms. Phantoms are based upon simple materials having biologic conductivity (< 5 S/m), including a cut of bone-in steak. The goal is not just to reconstruct an image that contains the features of the actual target, but also return correct conductivity values for the various features within the image.