Development of Selective Inhibitors of DNA Polymerase Delta: A Thesis

This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis. Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2\u27-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM. Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3\u27 → 5\u27 exonuclease activity of pol δ, displaying an IC50value greater than 1 mM. Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication. Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG–d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253–255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610–617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG–d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG–d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo

Temporary Electrostatic Impairment of DNA Recognition: Light‐Driven DNA Binding of Peptide Dimers

This is the peer reviewed version of the following article: Jiménez‐Balsa, A. , Pazos, E. , Martínez‐Albardonedo, B. , Mascareñas, J. L. and Vázquez, M. E. (2012), Temporary Electrostatic Impairment of DNA Recognition: Light‐Driven DNA Binding of Peptide Dimers. Angew. Chem. Int. Ed., 51: 8825-8829, which has been published in final form at https://doi.org/10.1002/anie.201201627. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsAppending negatively charged Glu8 tails to a peptide dimer derived from the GCN4 transcription factor leads to an effective suppression of its DNA binding. The specific DNA recognition can be restored by irradiation with UV light by using a photolabile linker between the acidic tail and the DNA binding peptideConsolider Ingenio. Grant Numbers: SAF2010‐20822‐C02, CTQ2009‐14431/BQU, CSD2007‐00006 Xunta de Galicia. Grant Numbers: INCITE09 209 084PR, PGIDIT08CSA‐047209PR, GRC2010/12 MINECOS

The ecology of exercise: mechanisms underlying Individual variation in behavior, activity, and performance: an introduction to symposium

Wild animals often engage in intense physical activity while performing tasks vital for their survival and reproduction associated with foraging, avoiding predators, fighting, providing parental care, and migrating. In this theme issue we consider how viewing these tasks as “exercise”—analogous to that performed by human athletes—may help provide insight into the mechanisms underlying individual variation in these types of behaviors and the importance of physical activity in an ecological context. In this article and throughout this issue, we focus on four key questions relevant to the study of behavioral ecology that may be addressed by studying wild animal behavior from the perspective of exercise physiology: (1) How hard do individual animals work in response to ecological (or evolutionary) demands?; (2) Do lab-based studies of activity provide good models for understanding activity in free-living animals and individual variation in traits?; (3) Can animals work too hard during “routine” activities?; and (4) Can paradigms of “exercise” and “training” be applied to free-living animals? Attempts to address these issues are currently being facilitated by rapid technological developments associated with physiological measurements and the remote tracking of wild animals, to provide mechanistic insights into the behavior of free-ranging animals at spatial and temporal scales that were previously impossible. We further suggest that viewing the behaviors of non-human animals in terms of the physical exercise performed will allow us to fully take advantage of these technological advances, draw from knowledge and conceptual frameworks already in use by human exercise physiologists, and identify key traits that constrain performance and generate variation in performance among individuals. It is our hope that, by highlighting mechanisms of behavior and performance, the articles in this issue will spur on further synergies between physiologists and ecologists, to take advantage of emerging cross-disciplinary perspectives and technologies

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study

<p>Abstract</p> <p>Background</p> <p>Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed.</p> <p>Results</p> <p>Our construct design strategy had four tactics: <it>N</it>- and <it>C</it>-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort.</p> <p>Conclusion</p> <p>Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.</p

Coupled Folding and Specific Binding: Fishing for Amphiphilicity

Proteins are uniquely capable of identifying targets with unparalleled selectivity, but, in addition to the precision of the binding phenomenon, nature has the ability to find its targets exceptionally quickly. Transcription factors for instance can bind to a specific sequence of nucleic acids from a soup of similar, but not identical DNA strands, on a timescale of seconds. This is only possible with the enhanced kinetics provided for by a natively disordered structure, where protein folding and binding are cooperative processes. The secondary structures of many proteins are disordered under physiological conditions. Subsequently, the disordered structures fold into ordered structures only when they bind to their specific targets. Induced folding of the protein has two key biological advantages. First, flexible unstructured domains can result in an intrinsic plasticity that allows them to accommodate targets of various size and shape. And, second, the dynamics of this folding process can result in enhanced binding kinetics. Several groups have hypothesized the acceleration of binding kinetics is due to induced folding where a “fly-casting” effect has been shown to break the diffusion-limited rate of binding. This review describes experimental results in rationally designed peptide systems where the folding is coupled to amphiphilicity and biomolecular activity

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia–ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns