Student Projects for UMass Chemistry 423 Spring 2012-9

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Phosphatase Inhibitor complexes: pdb 1nny

PTP1B inhibitor (PDB code 1nny)


Diabetes is a quickly growing disease that is affecting more and more people every day. Diabetes is caused when the body either does not produce enough insulin, or has a resistance to insulin. Insulin resistance occurs when an increase in the concentration of insulin in the cells leads to a decrease in the cell’s uptake of insulin.

When insulin binds to a normal insulin receptor, three specific tyrosine residues are phosphorylated and this serves as the first step in insulin signaling. The insulin receptor is bound to the cell by a beta strand that extends through the membrane into the interior of the cell. The insulin binds to the two different binding sites on the receptor and is used to move the glucose in the blood stream to different tissues in order to use as energy.

In a person with type 2 diabetes, the cells are unable to uptake glucose due to decreased insulin receptor signaling. This decrease in signaling can be caused by the protein tyrosine phosphatase (PTP1B). PTP1B is responsible for the dephosphorylation of the insulin receptor, and therefore responsible for the down regulation of insulin signaling and the cause of diabetes.

As explained below in the section labelled 'Binding Interactions,' recent efforts have been made to develop new drugs that would inhibit the effects of the PTP1B protein. A small, potent and selective inhibitor was developed that competitively and reversibly binds to the binding sites on the insulin receptor and selectively inhibits PTP1B. This PTP1B inhibitor increases the half-life of the phosphorylated insulin receptor, which in turn enhances the effects of insulin in patients with type 2 diabetes. [1]

PTP1B catalytic loop

Overall Structure

The secondary structure of the monomer includes 8 alpha and 10 beta strands, 8 beta strands make up the main . The beta sheet adopts a highly twisted configuration. The C-terminal are predominantly hydrophobic in nature and function in targeting the enzyme to the cytoplasmic face of membranes of the endoplasmic reticulum (ER). A structural feature that is highly conserved among PTPs is the catalytic, or PTP . The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. PTP loop is made up of the following residues (I/V)HCXAGXGR(S/T)G that includes the Cys215 . [2] Another conserved loop, the recognition loop, plays an important role in substrate recognition. The pTyr loop contains Tyr 46 (tyrosine), which defines the depth of the cleft and contributes to the specificity for phosphotyrosine-containing substrates. The Tyr 46 and Val 49 assist the substrate's insertion into catalytic site. 216 of the PTP loop forms a hydrogen bond with the the recognition loop, stabilizing the active site cleft. A third conserved loop is the WPD . For this specific inhibitor of the enzyme, once the substrate binds there is no conformational change in the structure of the loop.[1] For other inhibitors of this enzyme, once the substrate binds to the binding site, there is a conformational change in which the WPD loop closes around the side chain of the pTyr residue of the substrate. This causes Phe 282 to stack against the phenyl side chain of of the substrate pTyr, stabilizing the closed loop.[1] This conformation positions the Asp to function as a general acid in the initial part of the phosphorylation transfer step. PTP1B represents an example of the concept of an induced fit, which means that the binding of the substrate induces a conformational change that creates a form of the enzyme that is catalytic.

Binding Interactions

PTP1B is thought to primarily be responsible for the dephosphorylation of the insulin receptor and, therefore, acts to downregulate insulin signaling. Inhibiting PTP1B is linked to improved insulin response and activation of the insulin pathway. PTP1B deficient mice showed a resistance to diet induced diabetes.

Enzyme active site

Overall the is a competitive inhibitor which reversibly binds to two sites simultaneously and has a high selectivity for PTP1B over other phosphatases. This ligand was engineered by a research group from Abbot labs and the steps they took in its design revolve around many important binding interactions. The group designed the drug in . In order to identify this inhibiting ligand, the research group of interest first used a computer to scan a library of 10,000 organic compounds based on there NMR spectra, searching for those who have an affinity for the enzymes active site. The best match produced by the screen was diaryloxamic acid which the group then modified to naphthyloxamic acid , reasoning that an inhibitor that took up more space would be a more potent inhibitor. When the group mixed this molecule with enzyme, NMR data showed a chemical shift in , part of the active site of the enzyme. The observed kinetics indicated competitive and reversible inhibition. After their initial success, the group then reasoned that since the active domain of PTP1B is somewhat conserved by many other phosphatases and extremely similar in some ( TCPTP), incorporation of binding to a second and nonactive site would dramatically increase selectivity. Based off of naphthyloxamic acid, the group then created what they called compound 12 hoping that it would satisfy these conditions. Mixing the new ligand and enzyme then screening with NMR N-15 and C-13 resonance yielded data showing that in addition to binding to Val49, Gly220, and Gly 218, the new ligand bound to additional active sites, those being The new ligand also forms hydrogen bonds to the backbone of residues Ser216-Gly220 as well as forming a hydrophobic interaction with Tyr46. The group improved the site two ligand creating compound 23 compound 23, which primarily adds interactions with . It is not yet know whether Arg 24, Arg 254, or both residues are interacting with the ligand, but it is believed that they are either forming hydrogen bonds to the ligand or forming a salt bridge. The groups final ligand shows excellent potency for PTP1B while also showing high seletivity against other phosphatases.

Additional Features

Dephosphorylation reaction

Dephosphorylation of the Tyrosine-Phosphate Residue

A phosphorylated residue enters the active site of the protein, facilitated by the recognition loop residues . The base of the entrance through which it is brought is known as the PTP loop. The phosphotyrosine entering is amphipathic, thus requiring a non-polar pocket for its phenol ring while the polar end is positioned in the catalytic site. Once inside, the substrate causes a conformational change in the WPD loop, causing the substrate to be held in place for a nucleophilic attack. Also, the residue, , is shifted, so that it can act as an acid. The first step of the reaction involves Asp 181 attaching a hydrogen atom to the oxygen of tyrosine, thus neutralizing it and allowing it to diffuse from the site. Binding in the PTP loop occurs, so the connection between Arg 221 and the phosphate of the substrate is maximized. The WPD loop now has a very stable conformation, as binding has increased with multiple surrounding residues, . The tyrosine residue is now positioned in close proximity to the , allowing Cys 215 to remove the phosphate as an intermediate step in the reaction. Now, the phosphate binds to the , forming the cysteinyl-phosphate intermediate. This intermediate will then be dephosphorylated again, creating a water phosphate complex. Afterwards, the enzyme will return to its resting conformation and will be able to accept another tyrosine for dephosphorylation.[3]


Introduction - Jill Carlson

Overall Structure - Polina Berdnikova

Drug Binding Site - Brett Clinton

Additional Features - James Hamblin


  1. Szczepankiewicz BG, Liu G, Hajduk PJ, Abad-Zapatero C, Pei Z, Xin Z, Lubben TH, Trevillyan JM, Stashko MA, Ballaron SJ, Liang H, Huang F, Hutchins CW, Fesik SW, Jirousek MR. Discovery of a potent, selective protein tyrosine phosphatase 1B inhibitor using a linked-fragment strategy. J Am Chem Soc. 2003 Apr 9;125(14):4087-96. PMID:12670229 doi:10.1021/ja0296733
  2. Tonks NK. PTP1B: from the sidelines to the front lines! FEBS Lett. 2003 Jul 3;546(1):140-8. PMID:12829250

3. Edwards, K., T. Davis, D. Marcey, J. Kurihara, D. Yamamoto. 2001. Comparative Analysis of the Band 4.1/ezrin-related Protein Tyrosine Phosphatase Pez from Two Drosophila Species: Implication for Structure and Function. Gene 275: 195-205.

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