Henrik Sune Andersen

Structural and Evolutionary Relationships among Protein Tyrosine Phosphatase Domains

With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified—like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)—a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs.

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp181, Lys120, and Tyr46. PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Ligand-Induced Conformational Changes: Improved Predictions of Ligand Binding Conformations and Affinities

A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein tyrosine phosphatase 1B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal structure cocrystallized with a hexapeptide. The estimated binding energies for various docking modes as well as the RMS differences between the docked compounds and the crystallographic structure were calculated. In this scenario the estimated binding energies were not predictive inasmuch as docking modes with low estimated binding energies corresponded to relatively large RMS differences when aligned with the corresponding crystal structure. Secondly, the inhibitors were docked to their parent protein structures in which they were cocrystallized. In this case, there was a good correlation between low predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side chains exposed to the active site were considered in their allowed rotamer conformations and protein models containing all possible combinations of side-chain rotamers were generated. To evaluate which of these modeled active sites is the most likely binding site conformation for a certain inhibitor, the inhibitors were docked against all active site models. The receptor rotamer model corresponding to the lowest estimated binding energy is taken as the top candidate. Using this protocol, correct inhibitor binding modes could successfully be discriminated from proposed incorrect binding modes. Moreover, the ranking of the estimated ligand binding energies was in good agreement with experimentally observed binding affinities.

Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca2+-dependent exocytosis in pancreatic α- and β-cells

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at less than or equal to 2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC50) stimulation was observed at 24 muM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K+ channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca2+ current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC50 = 0.45 muM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC50 = I I nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K channel activity but resulted from a reduction of the rate of Ca2+-dependent exocytosis in single rat alpha-cells (EC50=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca2+-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules

A convenient large-scale chiral synthesis of protected 2-substituted 4-oxo-piperidine derivatives

Abstract A convenient large-scale chiral synthesis of protected 2-substituted-4-oxo-piperidine derivatives is described. Hetero Diels–Alder reaction between trifluoroacetic acid–boron trifluoride activated (1-phenyl-ethylimino)acetic acid ethyl ester and 2-trimethylsilyloxy-1,3-butadiene gave rise to a mixture of two diastereomers of 4-oxo-1-(1-phenyl-ethyl)-piperidine-2-carboxylic acid ethyl ester. Starting from (S)-1-phenyl-ethylamine pure adduct can be obtained by crystallization of the diastereomeric mixture. Reduction of the ester group gave rise to the corresponding hydroxymethyl analogue, which was subjected to further functional group transformations to yield the desired protected 2-aminomethyl-4-oxo-piperidine derivative without any racemization being observed.

A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant

Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.

Structure-Based Design of Protein Tyrosine Phosphatase Inhibitors

Protein tyrosine phosphatases (PTPs) are a family of intracellular enzymes that remove phosphate from tyrosine phosphorylated proteins. The PTP superfamily includes tyrosine phosphate-specific classical PTPs, dual-specificity PTPs, and low-molecularweight PTPs. PTPs and protein tyrosine kinases reversibly regulate the phosphotyrosine level in selected cellular proteins, thereby controlling many important signaling pathways in eukaryotes. Aberrant tyrosine phosphorylation levels have been associated with the development of cancer, autoimmunity, and diabetes, thus indicating that PTPs might play important etiological and pathogenic roles in these diseases. As a result, these enzymes have recently attracted much interest as potential drug targets. This is in particular due to the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity, thus indicating that PTP1B is an important negative regulator of insulin and leptin action and hence a potentially important drug target for the treatment of diabetes and obesity. The development of PTP inhibitors, in particular PTP1B inhibitors, has been greatly facilitated by an impressive number of X-ray structures that have allowed structure-based design of highly selective inhibitors of PTP1B, the main focus of this review. The initial attempts to design selective PTP inhibitors were based on replacement of pTyr with non-hydrolyzable phosphotyrosyl mimetics in small, efficient PTP peptide substrates, thereby utilizing both the potency and selectivity provided by the amino acid residues. However, several groups have now shown that it is possible to synthesize highly potent and selective non-phosphorus, non-peptide inhibitors of PTP1B. At this point, these achievements to some extent seem to have been reached at the expense of appropriate pharmacokinetic properties, including cellular uptake. Therefore, the next wave within the field of PTP inhibitors is likely to be focused on improvements in this respect. In addition, several other PTPs could potentially be attractive drug targets in autoimmunity and cancer.

A Facile Synthesis of Racemic 2-Aminomethyl-4-oxo-piperidine Intermediates

Abstract Facile access to the 2-substituted piperidine nucleus was achieved by a 3-component hetero Diels-Alder reaction. The use of an appropriately functionalized imine allowed the installation of a protecting group for both the piperidine and 2-position side chain nitrogens in one step. Also, by employing 2-trimethylsiloxy-1,3-butadiene, a reduction step was avoided, thus providing rapid entry into the piperidine core structure. This protocol led to the synthesis of some racemic 2-aminomethyl-4-oxo-piperidines; intermediates that were synthesized in multigram quantities.

Nonclinical pharmacokinetic and pharmacodynamic characterisation of somapacitan: A reversible non-covalent albumin-binding growth hormone

OBJECTIVE Somapacitan is an albumin-binding growth hormone derivative intended for once weekly administration, currently in clinical development for treatment of adult as well as juvenile GH deficiency. Nonclinical in vivo pharmacological characterisation of somapacitan was performed to support the clinical trials. Here we present the pharmacokinetic and pharmacodynamic effects of somapacitan in rats, minipigs, and cynomolgus monkeys. METHODS Pharmacokinetic studies investigating exposure, absorption, clearance, and bioavailability after single intravenous (i.v.) and subcutaneous (s.c.) administration were performed in all species. A dose-response study with five dose levels and a multiple dose pharmacodynamic study with four once weekly doses was performed in hypophysectomised rats to evaluate the effect of somapacitan on growth and IGF-I production. RESULTS Pharmacokinetic profiles indicated first order absorption from the subcutaneous tissue after s.c. injections for somapacitan in all three species. Apparent terminal half-lives were 5-6h in rats, 10-12h in minipigs, and 17-20h in monkeys. Somapacitan induced a dose-dependent growth in hypophysectomised rats (p<0.001) and an increase in plasma IGF-I levels in rats (p<0.01), minipigs (p<0.01), and cynomolgus monkeys (p<0.05) after single dose administration. Multiple once weekly dosing of somapacitan in hypophysectomised rats induced a step-wise increase in body weight with an initial linear phase the first 3-4days in each dosing interval (p<0.001). CONCLUSION The nonclinical pharmacokinetic and pharmacodynamic studies of somapacitan showed similar pharmacokinetic properties, with no absorption-limited elimination, increased clearance and increased and sustained levels of IGF-I in plasma for up to 10days after a single dose administration in all three species. Somapacitan induced a dose-dependent increase in body weight and IGF-I levels in hypophysectomised rats. Multiple dosing of somapacitan in hypophysectomised rats suggested a linear growth for the first 3-4days in each weekly dosing interval, whereas daily hGH dosing showed linear growth for approximately two weeks before reaching a plateau level.