Rob L. M. van Montfort

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.

Drugging the PI3 kinome: from chemical tools to drugs in the clinic.

The phosphatidylinositide 3-kinase (PI3K) pathway is very commonly activated in a wide range of human cancers and is a major driving force in oncogenesis. One of the class I lipid kinase members of the PI3K family, p110alpha, is probably the most commonly mutated kinase in the human genome. Alongside genetic, molecular biological, and biochemical studies, chemical inhibitors have been extremely helpful tools in understanding the role of PI3K enzymes in signal transduction and downstream physiological and pathological processes, and also in validating PI3Ks as therapeutic targets. Although they have been valuable in the past, the early and still frequently employed inhibitors, wortmannin and LY294002, have significant limitations as chemical tools. Here, we discuss the case history of the discovery and properties of an increasingly used chemical probe, the pan-class I PI3K and mammalian target of rapamycin (mTOR) inhibitor PI-103 (a pyridofuropyrimidine), and its very recent evolution into the thienopyrimidine drug GDC-0941, which exhibits excellent oral anticancer activity in preclinical models and is now undergoing phase I clinical trials in cancer patients. We also illustrate the impact of structural biology on the design of PI3K inhibitors and on the interpretation of their effects. The challenges and outlook for drugging the PI3 kinome are discussed in the more general context of the role of structural biology and chemical biology in innovative drug discovery.

Targeting HSP70 The second potentially druggable heat shock protein and molecular chaperone

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Structure-based design of molecular cancer therapeutics.

Structure-based approaches now impact across the whole continuum of drug discovery, from new target selection through the identification of hits to the optimization of lead compounds. Optimal application of structure-based design involves close integration with other discovery technologies, including fragment-based and virtual screening. Here, we illustrate the use of structural information and of structure-based drug design approaches in the discovery of small-molecule inhibitors for cancer drug targets and provide an outlook on the exploitation of structural information in future cancer drug discovery. Examples include high profile protein kinase targets and structurally related PI3 kinases, histone deacetylases, poly(ADP-ribose)polymerase and the molecular chaperone HSP90. Structure-based design approaches have also been successfully applied to the protein-protein interaction targets p53-MDM2 and the Bcl-2 family.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Insights into the Conformational Variability and Regulation of Human Nek2 Kinase

Summary The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-Å resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target.

The structure of the Escherichia coli phosphotransferase IIAmannitol reveals a novel fold with two conformations of the active site.

BACKGROUND The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates. Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways. The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II (EII). EIIs are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate. Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity. RESULTS We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIAmtl (M(r) 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIAmtl. The structure was refined at 1.8 A resolution to an R factor of 19.0% (Rfree 24.2%). The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues. The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIAmtl molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIAmtl complex were generated. CONCLUSIONS The fold of IIAmtl is completely different from the structures of other IIA proteins determined so far. The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIAmtl is involved. A comparison of the HPr-IIAmtl model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar. Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium.

The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases

BACKGROUND . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

Structure-Guided Evolution of Potent and Selective CHK1 Inhibitors through Scaffold Morphing

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.