Allosteric Modulation of Phosphatase Activity May Redefine

Aug 19, 2016 - Brodsky, J. L.; Zuiderweg, E. R. P.; Gestwicki, J. E. Binding of a small molecule at a protein-protein interface regulates the chaperon...
0 downloads 4 Views 1MB Size
Viewpoint pubs.acs.org/jmc

Allosteric Modulation of Phosphatase Activity May Redefine Therapeutic Value Joseph M. Salamoun and Peter Wipf* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States ABSTRACT: Despite their extensive involvement in signaling pathways and disease pathologies, targeting protein phosphatases remains an underexplored opportunity in drug discovery. Selective intracellular regulation of phosphatases with small molecule inhibitors has been an unmet challenge. However, recent progress in the development of allosteric modulators encourages renewed efforts to exploit their potential as therapeutic targets. especially in regard to finding allosteric inhibitors. Garcia Fortanet et al. began their search for an allosteric inhibitor of SHP2 by screening a 100000-compound library against a fulllength construct of SHP2.7 Nine hundred hit compounds were initially identified, with 30% or greater inhibition of the enzyme. The use of a truncated construct of SHP2 containing only the PTP domain (the catalytic site), in a control assay along with two other assays, allowed them to deprioritize any hits that targeted the phosphate binding site. The list of compounds was narrowed down to six, with SHP836 (1) becoming the lead structure. A cocrystal X-ray structure of 1 with SHP2 showed the molecule binding to a tunnel-like region at the intersection of the Cterminal SH2, N-terminal SH2, and PTP domains (Figure 1). Interestingly, the binding of pyrimidine 1 stabilizes the protein complex in its autoinhibitory conformation, mimicking the unactivated apo-SHP2, where access to the catalytic site is hindered by an intramolecular interaction with the N-terminal SH2 domain.8 This result highlights the utility of an allosteric site for modulating protein activity, mirroring an intrinsic process for phosphatase regulation. In addition to confirming allosteric binding, the X-ray structure allowed for the synthetic tailoring of analogues to bind in the specific pocket of interest, improving their potency and selectivity. Structure−activity relationship studies yielded a potent second-generation compound 2, SHP099, with an IC50 of 70 nM against SHP2 (Figure 2). The design of pyrazine 2 took advantage of specific amino acid interactions on the target enzyme, and a panel of 21 human phosphatases and 66 kinases confirmed its selectivity. Furthermore, this compound is highly soluble, orally bioavailable, and displayed dose-dependent tumor inhibition in a human esophageal squamous cell carcinoma KYSE-520 xenograft model with tumor stasis in mice achieved at a dose of 100 mg/kg. Allosteric inhibition is increasingly applied toward difficult drug targets such as chaperones and kinesins9,10 as well as forming an alternative drug development strategy for traditional active-site targets such as proteases and kinases. Cryo-electron microscopy is an emerging tool to analyze changes in protein dynamics upon interactions with allosteric inhibitors.11 The

T

he crucial role of phosphatases in regulating cellular functions is well documented.1 However, the druggability of this ubiquitous enzyme family has frequently been questioned.2 Phosphate group mimicry is notoriously difficult, and the phosphate group binding site is highly positively charged and generally lacks a distinct small molecule pocket. Further compounding the challenge for the medicinal chemist are the apparent need for phosphatase activators to oppose the action of kinase upregulation, the wide range of regulatory domains in phosphatases that are distinct from their catalytic domains, and assay paradigms that favor redox-active compounds and lipophilic “frequent hitters” binding to exposed hydrophobic surfaces of protein constructs.3 As a result, this class of essential proteins has been largely shunned in drug discovery, in contrast to protein kinases, which have yielded numerous marketed drugs.4 Compared to the state of the art in kinase drug discovery, the medicinal chemistry of phosphatase targeting will require at least one to two decades to catch up. Unlike kinases, phosphatases do not have a defined substrate like adenosine triphosphate (ATP) to guide drug design. While some therapeutic agents that modulate phosphatase activity are currently in use, these compounds were not developed via a target-oriented medicinal chemistry campaign. Instead, their activity against phosphatases was discovered after their regulatory approval. Eukaryotic cells contain about 100 phosphatases with vastly different functions that can be selectively turned on and off. One method of regulating protein catalytic activity is inducing conformational changes by targeting distinct allosteric sites through protein−protein interactions and the binding of cofactors.5 In a similar fashion, small molecules binding to specific allosteric pockets can alter a protein’s dynamics and conformational state, opening or closing access to the catalytic site. Allosteric pockets provide an opportunity for tailoring modulators with greater target selectivity, thus minimizing offtarget effects and potentially addressing one of the perceived short-comings of phosphatases as therapeutic targets. In this issue of the Journal of Medicinal Chemistry, Garcia Fortanet et al. report the development of such a highly potent, selective, and in vivo active allosteric inhibitor of SHP2, a nonreceptor protein tyrosine phosphatase of great therapeutic interest.6 Prioritizing hit compounds from a high-throughput screen can be a daunting process without the proper structural filters, © XXXX American Chemical Society

Received: August 10, 2016

A

DOI: 10.1021/acs.jmedchem.6b01210 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Viewpoint

Figure 1. (A) Crystal structure of unactivated apo-SHP2 in its autoinhibitory conformation; the catalytic site residues HCSAGIGRT are covered in a tight interface between the PTP and SH2 domains (PDB code 2SHP). Shown in blue is the PTP domain containing the catalytic site; the SH2 domains are colored red. (B) Co-crystal structure of compound 1 (green) bound to SHP2 and stabilizing a conformation that strongly resembles the autoinhibited structure (PDB code 5EHP). (6) Garcia Fortanet, J.; Chen, C. H.-T.; Chen, Y.-N. P.; Chen, Z.; Deng, Z.; Firestone, B.; Fekkes, P.; Fodor, M.; Fortin, P. D.; Fridrich, C.; Grunenfelder, D.; Ho, S.; Kang, Z. B.; Karki, R.; Kato, M.; Keen, N.; LaBonte, L. R.; Larrow, J.; Lenoir, F.; Liu, G.; Liu, S.; Lombardo, F.; Majumdar, D.; Meyer, M. J.; Palermo, M.; Perez, L.; Pu, M.; Ramsey, T.; Sellers, W. R.; Shultz, M. D.; Stams, T.; Towler, C.; Wang, P.; Williams, S. L.; Zhang, J.-H.; LaMarche, M. J. Allosteric inhibition of SHP2: Identification of a potent, selective, and orally efficacious phosphatase inhibitor. J. Med. Chem. 2016, DOI: 10.1021/acs.jmedchem.6b00680. (7) Chen, Y.-N. P.; LaMarche, M. J.; Chan, H. M.; Fekkes, P.; GarciaFortanet, J.; Acker, M. G.; Antonakos, B.; Chen, C. H.-T.; Chen, Z.; Cooke, V. G.; Dobson, J. R.; Deng, Z.; Fei, F.; Firestone, B.; Fodor, M.; Fridrich, C.; Gao, H.; Grunenfelder, D.; Hao, H.-X.; Jacob, J.; Ho, S.; Hsiao, K.; Kang, Z. B.; Karki, R.; Kato, M.; Larrow, J.; La Bonte, L. R.; Lenoir, F.; Liu, G.; Liu, S.; Majumdar, D.; Meyer, M. J.; Palermo, M.; Perez, L.; Pu, M.; Price, E.; Quinn, C.; Shakya, S.; Shultz, M. D.; Slisz, J.; Venkatesan, K.; Wang, P.; Warmuth, M.; Williams, S.; Yang, G.; Yuan, J.; Zhang, J.-H.; Zhu, P.; Ramsey, T.; Keen, N. J.; Sellers, W. R.; Stams, T.; Fortin, P. D. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 2016, 535, 148−152. (8) Hof, P.; Pluskey, S.; Dhe-Paganon, S.; Eck, M. J.; Shoelson, S. E. Crystal Structure of the tyrosine phosphatase SHP-2. Cell 1998, 92, 441−450. (9) Wisen, S.; Bertelsen, E. B.; Thompson, A. D.; Patury, S.; Ung, P.; Chang, L.; Evans, C. G.; Walter, G. M.; Wipf, P.; Carlson, H. A.; Brodsky, J. L.; Zuiderweg, E. R. P.; Gestwicki, J. E. Binding of a small molecule at a protein-protein interface regulates the chaperone activity of Hsp70-Hsp40. ACS Chem. Biol. 2010, 5, 611−622. (10) Yokoyama, H.; Sawada, J.-i.; Katoh, S.; Matsuno, K.; Ogo, N.; Ishikawa, Y.; Hashimoto, H.; Fujii, S.; Asai, A. Structural basis of new allosteric inhibition in kinesin spindle protein Eg5. ACS Chem. Biol. 2015, 10, 1128−1136. (11) Banerjee, S.; Bartesaghi, A.; Merk, A.; Rao, P.; Bulfer, S. L.; Yan, Y.; Green, N.; Mroczkowski, B.; Neitz, R. J.; Wipf, P.; Falconieri, V.; Deshaies, R. J.; Milne, J. L. S.; Huryn, D.; Arkin, M.; Subramaniam, S. 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 2016, 351, 871−875. (12) Lazo, J. S.; Sharlow, E. R. Drugging undruggable molecular cancer targets. Annu. Rev. Pharmacol. Toxicol. 2016, 56, 23−40. (13) Her, N.-G.; Toth, J. I.; Ma, C.-T.; Wei, Y.; Motamedchaboki, K.; Sergienko, E.; Petroski, M. D. p97 Composition changes caused by allosteric inhibition are suppressed by an on-target mechanism that increases the enzyme’s ATPase activity. Cell Chem. Biol. 2016, 23, 517− 528.

Figure 2. Hit compound 1 was optimized to yield the more potent analogue 2.

SHP2 inhibitor 2 joins a group of allosteric modulators of phosphatase targets that include DUSP6, WIP1, PTP1B, TCPTP, and Eya2.12 This growing portfolio provides a proof of concept for the feasibility of selectively targeting allosteric sites on phosphatases with drug-like small molecules. The ultimate test remains in the progression of these phosphatase modulators through clinical trials and FDA approval. A possible complication might be that allosteric inhibition is more readily overcome by target mutations than orthosteric inhibition.13 If allosteric inhibitors successfully pass the clinical trial stage, it is likely that a surge in drug discovery on phosphatases will result, similar to the way that the approval of imatinib influenced the development of drugs acting on protein kinases.

■ ■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 412-624-8606.

REFERENCES

(1) Hunter, T. Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling. Cell 1995, 80, 225−236. (2) De Munter, S.; Köhn, M.; Bollen, M. Challenges and opportunities in the development of protein phosphatase-directed therapeutics. ACS Chem. Biol. 2013, 8, 36−45. (3) Lazo, J. S.; Wipf, P. Phosphatases as targets for cancer treatment. Curr. Opin. Invest. Drugs 2009, 10, 1297−1304. (4) Roskoski, R. A historical overview of protein kinases and their targeted small molecule inhibitors. Pharmacol. Res. 2015, 100, 1−23. (5) Motlagh, H. N.; Wrabl, J. O.; Li, J.; Hilser, V. J. The ensemble nature of allostery. Nature 2014, 508, 331−339. B

DOI: 10.1021/acs.jmedchem.6b01210 J. Med. Chem. XXXX, XXX, XXX−XXX