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The P(2)' residue is a key determinant of mesotrypsin specificity: engineering a high-affinity inhibitor with anticancer activity.

Salameh MA, Soares AS, Hockla A, Radisky DC, Radisky ES
Biochem J 440 95-105 (2011)
Cited: 31 times
EuropePMC logo PMID: 21806544

Abstract

PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

Reviews - 3p95 mentioned but not cited (1)

  1. Biochemical and structural insights into mesotrypsin: an unusual human trypsin. Salameh MA, Radisky ES. Int J Biochem Mol Biol 4 129-139 (2013)

Articles - 3p95 mentioned but not cited (7)

  1. The P(2)' residue is a key determinant of mesotrypsin specificity: engineering a high-affinity inhibitor with anticancer activity. Salameh MA, Soares AS, Hockla A, Radisky DC, Radisky ES. Biochem J 440 95-105 (2011)
  2. Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates. Alloy AP, Kayode O, Wang R, Hockla A, Soares AS, Radisky ES. J Biol Chem 290 21523-21535 (2015)
  3. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis. Kayode O, Wang R, Pendlebury DF, Cohen I, Henin RD, Hockla A, Soares AS, Papo N, Caulfield TR, Radisky ES. J Biol Chem 291 26304-26319 (2016)
  4. Ligand-target prediction by structural network biology using nAnnoLyze. Martínez-Jiménez F, Marti-Renom MA. PLoS Comput Biol 11 e1004157 (2015)
  5. Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin. Cohen I, Coban M, Shahar A, Sankaran B, Hockla A, Lacham S, Caulfield TR, Radisky ES, Papo N. J Biol Chem 294 5105-5120 (2019)
  6. Small molecule inhibitors of mesotrypsin from a structure-based docking screen. Kayode O, Huang Z, Soares AS, Caulfield TR, Dong Z, Bode AM, Radisky ES. PLoS One 12 e0176694 (2017)
  7. Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks. van den Kerkhof DL, Nagy M, Wichapong K, Brouns SLN, Heemskerk JWM, Hackeng TM, Dijkgraaf I. Res Pract Thromb Haemost 5 231-242 (2021)


Reviews citing this publication (2)

  1. Recent advances on plasmin inhibitors for the treatment of fibrinolysis-related disorders. Al-Horani RA, Desai UR. Med Res Rev 34 1168-1216 (2014)
  2. Function and clinical relevance of kallikrein-related peptidases and other serine proteases in gynecological cancers. Dorn J, Beaufort N, Schmitt M, Diamandis EP, Goettig P, Magdolen V. Crit Rev Clin Lab Sci 51 63-84 (2014)

Articles citing this publication (21)

  1. PRSS3/mesotrypsin is a therapeutic target for metastatic prostate cancer. Hockla A, Miller E, Salameh MA, Copland JA, Radisky DC, Radisky ES. Mol Cancer Res 10 1555-1566 (2012)
  2. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms. Chen Z, Wang B, Hu J, Yang W, Cao Z, Zhuo R, Li W, Wu Y. PLoS One 8 e57529 (2013)
  3. PEGylation extends circulation half-life while preserving in vitro and in vivo activity of tissue inhibitor of metalloproteinases-1 (TIMP-1). Batra J, Robinson J, Mehner C, Hockla A, Miller E, Radisky DC, Radisky ES. PLoS One 7 e50028 (2012)
  4. Sequence and conformational specificity in substrate recognition: several human Kunitz protease inhibitor domains are specific substrates of mesotrypsin. Pendlebury D, Wang R, Henin RD, Hockla A, Soares AS, Madden BJ, Kazanov MD, Radisky ES. J Biol Chem 289 32783-32797 (2014)
  5. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy. Cohen I, Kayode O, Hockla A, Sankaran B, Radisky DC, Radisky ES, Papo N. Biochem J 473 1329-1341 (2016)
  6. PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma. Ma H, Hockla A, Mehner C, Coban M, Papo N, Radisky DC, Radisky ES. Sci Rep 9 1844 (2019)
  7. Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors. Salameh MA, Soares AS, Alloy A, Radisky ES. Protein Sci 21 1103-1112 (2012)
  8. A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering. Sananes A, Cohen I, Shahar A, Hockla A, De Vita E, Miller AK, Radisky ES, Papo N. J Biol Chem 293 12663-12680 (2018)
  9. Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis. Szabó A, Ludwig M, Hegyi E, Szépeová R, Witt H, Sahin-Tóth M. J Biol Chem 290 17282-17292 (2015)
  10. Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme. García-Fernández R, Perbandt M, Rehders D, Ziegelmüller P, Piganeau N, Hahn U, Betzel C, Chávez Mde L, Redecke L. J Biol Chem 290 14154-14165 (2015)
  11. Functional characterization of a new non-Kunitz serine protease inhibitor from the scorpion Lychas mucronatus. Liu H, Chen J, Wang X, Yan S, Xu Y, San M, Tang W, Yang F, Cao Z, Li W, Wu Y, Chen Z. Int J Biol Macromol 72 158-162 (2015)
  12. Specificity profiling of human trypsin-isoenzymes. Schilling O, Biniossek ML, Mayer B, Elsässer B, Brandstetter H, Goettig P, Stenman UH, Koistinen H. Biol Chem 399 997-1007 (2018)
  13. Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries. Naftaly S, Cohen I, Shahar A, Hockla A, Radisky ES, Papo N. Nat Commun 9 3935 (2018)
  14. PRSS3/mesotrypsin in prostate cancer progression: implications for translational medicine. Radisky ES. Asian J Androl 15 439-440 (2013)
  15. Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases. Cohen I, Naftaly S, Ben-Zeev E, Hockla A, Radisky ES, Papo N. Biochem J 475 1335-1352 (2018)
  16. Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes. Heyne M, Shirian J, Cohen I, Peleg Y, Radisky ES, Papo N, Shifman JM. J Am Chem Soc 143 17261-17275 (2021)
  17. Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA. Borovsky D, Hancock RG, Rougé P, Powell CA, Shatters RG. Arch Insect Biochem Physiol 99 e21506 (2018)
  18. SAXS Analysis and Characterization of Anticancer Activity of PNP-UDP Family Protein from Putranjiva roxburghii. Verma P, Varshney R, Yadav SPS, Kar B, Roy P, Sharma AK. Protein J 41 381-393 (2022)
  19. Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site. Lacham-Hartman S, Shmidov Y, Radisky ES, Bitton R, Lukatsky DB, Papo N. PLoS One 16 e0249616 (2021)
  20. Serine Protease 3 Promotes Progression of Diffuse Large B-Cell Lymphoma and Serves as a Novel Prognostic Predictor. Zheng L, Wang X, Zheng W, Huang H. Dis Markers 2022 1254790 (2022)
  21. Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers. Sananes A, Cohen I, Allon I, Ben-David O, Abu Shareb R, Yegodayev KM, Stepensky D, Elkabets M, Papo N. Mol Oncol 17 2337-2355 (2023)


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