Type 2 Diabetes Reversal

Paper in Nature Chemical Biology

Understanding the basis and treatment of disease.

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Type 2 Diabetes Reversal

#1  Postby Calilasseia » Mar 28, 2017 6:35 pm

New Scientist reports on a possibly ground breaking development here:

First steps toward a medication that could reverse Type 2 Diabetes

The paper cited is this one:

Diabetes Reversal By Inhibition Of The Low-Molecular Weight Tyrosine Phosphatase by Stephanie M Stanford, Alexander E Aleshin, Vida Zhang, Robert J Ardecky, Michael P Hedrick, Jiwen Zou, Santhi R Ganji, Matthew R Bliss, Fusayo Yamamoto, Andrey A Bobkov, Janna Kiselar, Yingge Liu, Gregory W Cadwell, Shilpi Khare, Jinghua Yu, Antonio Barquilla, Thomas D Y Chung, Tomas Mustelin, Simon Schenk, Laurie A Bankston, Robert C Liddington, Anthony B Pinkerton & Nunzio Bottini, Nature Chemical Biology, DOI: 10.1038/nchembio.2344

Stanford et al, 2017 wrote:Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.

From the paper in more detail:

Stanford et al, 2017 wrote:Identification of a novel small-molecule LMPTP inhibitor

We sought to explore the role of LMPTP in insulin signaling using a small-molecule inhibitor; thus, we embarked on a high-throughput screening (HTS) campaign. Previous searches for PTP inhibitors targeted the highly charged active site, typically yielding compounds with high activity in vitro but low selectivity and/or cellular permeability16. To disfavor competitive inhibition, we employed a high concentration of substrate (0.4 mM 3-O-methylfluorescein phosphate; OMFP) such that the enzyme was at Vmax. We also included detergent (0.01% Triton X-100) to minimize compound aggregation and false-positive hits25 . The screening conditions are described in Supplementary Table 1.

Our HTS workflow (Supplementary Fig. 6) started with 364,168 small-molecule compounds from the National Institutes of Health Molecular Libraries Small Molecule Repository. Using a primary screen, cheminformatic filtering of “pan-assay interference compounds” (PAINS)26 , dose–response assays with OMFP and paranitrophenylphosphate (pNPP) substrates, and counterscreens against class I tyrosine-specific lymphoid phosphatase (LYP) and class I dual-specific vaccinia H1-related phosphatase (VHR), we identified 17 hits. Retesting fresh powders against LMPTP, LYP and VHR revealed three confirmed hits with different scaffolds Fig. 2a): MLS-0045954 (1), MLS-0251308 (2) and MLS-0322825 (3).

We selected 3 for further study based on potency and lack of promiscuity in a PubChem analysis. Dose–response assays for 3 are shown in Figure 2b.

From the discussion:

Standford et al, 2017 wrote:DISCUSSION

Obesity is frequently complicated by insulin resistance and type 2 diabetes1,2 . Development of insulin-sensitizing agents to minimize the need for injectable insulin remains a major unmet medical need. LMPTP has been proposed to inhibit insulin signaling, and human genetics studies suggest that high LMPTP activity promotes diabetes. We report that LMPTP promotes high-fat diet-induced insulin resistance and diabetes through an action on the liver. We found that LMPTP deletion enhanced insulin-induced liver IR signaling, suggesting that LMPTP acts by suppressing IR activation in the liver. This observation is supported by evidence that chemical inhibition of LMPTP activity increases insulin-induced activation of the liver IR and downstream signaling pathways and improves glucose tolerance in obese mice.

Our study exemplifies the importance of discovering specific PTP inhibitors with in vivo activity to firmly establish the physiological role of PTPs and their potential as drug targets. Substantial progress has been made toward increasing the potency of chemical LMPTP inhibitors18–22 . However, the lack of selectivity of published compounds has limited their usefulness in studying LMPTP functions in vivo. Our new LMPTP inhibitors display excellent drug-like properties and represent strong leads for further optimization.

Although our inhibitors bound LMPTP at the opening of the active site pocket, unlike the majority of previous phosphatase inhibitors16,17, they lack a phosphate-mimic active site binding group. Data derived from enzymatic, biophysical and mutagenesis studies on this series are consistent with an uncompetitive mode of LMPTP inhibition. These compounds do not compete for binding with a phosphotyrosine substrate; rather, they bind most tightly to the phosphocysteine intermediate, mimicked here by the LMPTP– vanadate complex. They fully occupy the pocket above the LMPTP active site, excluding access to water required for the final hydrolysis step in catalysis. Early NMR studies elegantly described a preformed active site for LMPTP, demonstrating that while the apo enzyme showed some disorder in the N-terminal helix, saturating levels of phosphate caused a local disorder-to-order transition in the helix36 . Addition of vanadate produced an identical effect on the helix, and no other effects on the structure beyond the active site cysteine and its immediate environment36 . In our ITC experiments, binding of 18 to LMPTP was enthalpy driven when vanadate was prebound but entropy driven in its absence. This suggests that vanadate binding creates a more rigid environment that enables 18 to bind in a unique conformation, which contributes to its specific binding.

Our structural data allow us to formulate a rationale for the uncompetitive nature of these inhibitors. Although they can bind to the free enzyme with modest affinity, binding of the free enzyme to negatively charged phosphotyrosine substrate is preferred, owing to favorable electrostatic interactions conferred by the positive charge on R18 and the positive end of the helix 1 dipole. Following nucleophilic attack on the substrate and cleavage of the phosphotyrosine bond, a negatively charged phosphocysteine (S-PO32−) intermediate is created, which neutralizes the positive charge in the pocket. This configuration facilitates inhibitor binding, which is dominated by aromatic and/or hydrophobic interactions with the deep front and back of the pocket and anchored on two sides by electrostatic interactions. The inhibitor fills the active site cavity completely, forming a ‘hand-in-glove’ water-tight seal. The S-PO32− moiety lacks an axial phosphate oxygen, enabling the edge of the quinoline ring to approach closely and ensure steric occlusion of water. The hydrogen bond between the quinoline ring and D129 prevents hydrolysis while the inhibitor is bound to the enyzme. The benzonitrile ring attached to the quinoline ring provides further anchorage and steric occlusion of water at this side of the pocket.

In conclusion, we performed the first characterization of the metabolic role of LMPTP and report discovery of a selective LMPTP inhibitor series and the first orally bioavailable LMPTP inhibitor. Although further studies on LMPTP in insulin signaling are warranted, our data establish LMPTP as a key promoter of obesity-induced diabetes and drug target for obesity-associated insulin resistance. Our LMPTP inhibitor will be instrumental for further understanding the biology of LMPTP in vivo. We envision that the information we collected about its mechanism of action and binding mode will pave the way for development of LMPTP inhibitors suitable for therapeutic testing in human diabetes. Since LMPTP has also been proposed to promote heart failure23 and tumor growth37, such inhibitors are predicted to have a wide range of therapeutic applications.

Basically, the authors found that the LMPTP enzyme is implicated in loss of insulin receptor sensitivity, and that selective inhibition of this enzyme can not only bring type 2 diabetes to a halt in the mouse model, but reverse it. The move to find clinically safe variations of the inhibitors tested in the mouse model, capable of being deployed in humans, is now underway.
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