Dominic Tisi

Oxidation State of the Active-Site Cysteine in Protein Tyrosine Phosphatase 1B

Protein tyrosine phosphatases regulate signal transduction pathways involving tyrosine phosphorylation and have been implicated in the development of cancer, diabetes, rheumatoid arthritis and hypertension. Increasing evidence suggests that the cellular redox state is involved in regulating tyrosine phosphatase activity through the reversible oxidization of the catalytic cysteine to sulphenic acid (Cys-SOH). But how further oxidation to the irreversible sulphinic (Cys-SO2H) and sulphonic (Cys-SO3H) forms is prevented remains unclear. Here we report the crystal structures of the regulatory sulphenic and irreversible sulphinic and sulphonic acids of protein tyrosine phosphatase 1B (PTP1B), an important enzyme in the negative regulation of the insulin receptor and a therapeutic target in type II diabetes and obesity. We also identify a sulphenyl-amide species that is formed through oxidation of its catalytic cysteine. Formation of the sulphenyl-amide causes large changes in the PTP1B active site, which are reversible by reduction with the cellular reducing agent glutathione. The sulphenyl-amide is a protective intermediate in the oxidative inhibition of PTP1B. In addition, it may facilitate reactivation of PTP1B by biological thiols and signal a unique state of the protein.

Recent Developments in Fragment-Based Drug Discovery

The field of fragment-based drug discovery (FBDD) has developed significantly over the past 10 years and is now recognized as a tangible alternative to more traditional methods of hit identification, such as high throughput screening (HTS). The number of commercial and academic groups actively engaged in fragment-based research has increased, and as a consequence, there has been continued development and refinement of techniques and methods. From its inception, the fragment-based approach had two central tenets that were critical to its success and that have set it apart from HTS and other hit identification techniques. The first is the concept that chemical space can be more efficiently probed by screening collections of small fragments rather than libraries of larger molecules. The number of potential fragments with up to 12 heavy atoms (not including threeand four-membered ring structures) has been estimated at 10, whereas the number of potential druglike molecules with up to 30 heavy atoms is estimated at more than 10. Therefore, a much greater proportion of “fragment-like” chemical space can feasibly be screened in FBDD compared to “druglike” chemical space covered in a HTS where molecular size is much larger. The second idea is that, because by definition fragment molecules are small in size (typically less than 250 Da), they should typically bind with lower affinity to their target protein (micromolar to millimolar range) compared with druglike molecules that can form many more interactions (nanomolar to micromolar range) but that the binding efficiency per atom is at least as high as for larger hit molecules. Implicitly, the screening techniques employed in FBDD must be correspondingly much more sensitive than a HTS bioassay. Generally, sensitive biophysical techniques are employed to detect these weak binding events and to characterize the fragment interactions with the target active site. Nuclear magnetic resonance (NMR) and protein X-ray crystallography have been used extensively in fragment-based research because these techniques are highly sensitive in detecting low affinity fragment binding and also give information about the fragmentprotein interactions being formed. There have been a number of recently published general review articles that have discussed the various aspects of the FBDD field. In addition, there are now two books on the subject. In this journal in 2004, Erlanson et al. summarized the major developments in FBDD since the original publication by Fesik and co-workers of the “SAR by NMR” approach in the late 1990s. Particular note was given to the biophysical methods employed to screen for fragment binding and the merits and drawbacks of each of these techniques, along with the approaches that can be used to optimize fragments into lead molecules. Herein, the trends and developments over the past 4 years will be outlined and some selected examples that are illustrative of the approaches being utilized by those active in the field examined. Additionally, this review will look in some detail at representative protein-ligand complexes observed between fragment-sized molecules and their protein targets from the Protein Data Bank (PDB). Finally, some conclusions will be drawn from these data and the future of FBDD discussed.

Fragment-Based Discovery of the Pyrazol-4-Yl Urea (at9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity.

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

Structural biology and anticancer drug design

The discovery and development of a new drug are parts of a multistep process that starts with the detailed analysis of the disease to be investigated. The next step is the identification of the key biological molecules that are involved in the disease process whereby modulation of these molecules, usually proteins, will likely have a therapeutic effect. Using recombinant DNA technology, these molecules can be produced and functional assays developed to monitor the level of activity in the presence of potential inhibitors. The screening stage of the drug discovery process is a continually evolving field with new technologies being developed to improve the speed, accuracy, and sensitivity of detecting compounds that bind to the target protein (“hits”). Traditionally, functional assays have been used as the primary identifier of compound hits. Over the past decade, however, high-throughput protein crystallography has continued to evolve as a pivotal technique in early hit generation.

Engineering and purification of a thermostable, high-yield, variant of PfCRT, the Plasmodium falciparum chloroquine resistance transporter

Historically chloroquine was used to treat the most deadly form of malaria, caused by the parasite Plasmodium falciparum. The selective pressure of chloroquine therapy led to the rapid emergence of chloroquine resistant parasites. Resistance has been attributed to the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT), an integral membrane protein of unknown structure. A PfCRT structure would provide new insights into how the protein confers chloroquine resistance and thereby also yield novel opportunities for developing anti-malarial therapies. Although PfCRT is an attractive target for characterisation and structure determination, very little work has been published on its expression and purification. Here we present a medium throughput protocol, employing Sf9 insect cells, for testing the expression, stability and purification yield of rationally designed PfCRT mutant constructs and constructs of a PfCRT orthologue from Neospora caninum (NcCRT). We have identified a conserved cysteine residue in PfCRT that results in elevated protein stability when mutated. Combining this mutation with the insertion of T4-lysozyme into a specific surface loop further augments PfCRT protein yield and thermostability. Screening also identified an NcCRT construct with an elevated purification yield. Furthermore it was possible to purify both PfCRT and NcCRT constructs at milligram-scales, with high purities and with size exclusion chromatography profiles that were consistent with monodispersed, homogeneous protein.

CHAPTER 4 – Structural biology and anticancer drug design

Publisher Summary This chapter discusses the structural biology and anticancer drug design. The use of structural biology techniques, such as NMR and X-ray crystallography, are now widely accepted to be powerful tools in structure-based drug design. The discovery and development of a new drug is a multi-step process, which starts with the detailed analysis of the disease to be investigated. The next step is the identification of the key biological molecules that are involved in the disease process, whereby modulation of these molecules, usually proteins, will likely have a therapeutic effect. Particularly in the case of X-ray crystallography, the advent of improved equipment, software, and techniques has resulted in a dramatic improvement in the speed with which structural data can be obtained. Structural biology methods enable the generation of a three-dimensional model of a protein molecule by various techniques, allowing researchers to gain powerful insights into the structure and mechanisms by which proteins and larger protein complexes operate. Structural biology now has an impact throughout the drug discovery process, from hit identification through to late-stage lead optimization. The structure-guided design of AT7519 provides a good example of this being put into practice for an oncology target.