Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
22 May 2024
22 May 2024
Historique:
received:
28
09
2023
accepted:
09
05
2024
medline:
23
5
2024
pubmed:
23
5
2024
entrez:
22
5
2024
Statut:
epublish
Résumé
Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.
Identifiants
pubmed: 38777835
doi: 10.1038/s41467-024-48655-w
pii: 10.1038/s41467-024-48655-w
doi:
Substances chimiques
High-Temperature Requirement A Serine Peptidase 1
EC 3.4.21.-
HTRA1 protein, human
EC 3.4.21.-
Peptides
0
Peptide Library
0
Cystine
48TCX9A1VT
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
4359Informations de copyright
© 2024. The Author(s).
Références
Colgrave, M. L. & Craik, D. J. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43, 5965–5975 (2004).
pubmed: 15147180
doi: 10.1021/bi049711q
Gao, X. et al. Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking. Sci. Rep. 6, 35179 (2016).
pubmed: 27734922
pmcid: 5062073
doi: 10.1038/srep35179
Stanger, K. et al. Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A. FEBS Lett. 588, 4487–4496 (2014).
pubmed: 25448598
doi: 10.1016/j.febslet.2014.10.020
Daly, N. L. & Craik, D. J. Bioactive cystine knot proteins. Curr. Opin. Chem. Biol. 15, 362–368 (2011).
pubmed: 21362584
doi: 10.1016/j.cbpa.2011.02.008
Kintzing, J. R. & Cochran, J. R. Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles. Curr. Opin. Chem. Biol. 34, 143–150 (2016).
pubmed: 27642714
doi: 10.1016/j.cbpa.2016.08.022
Heitz, A., Chiche, L., Le-Nguyen, D. & Castro, B. 1H 2D NMR and distance geometry study of the folding of Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family. Biochemistry 28, 2392–2398 (1989).
pubmed: 2730872
doi: 10.1021/bi00432a009
Hansen, S. et al. Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/beta-catenin signaling. Proc. Natl Acad. Sci. USA 119, e2207327119 (2022).
pubmed: 36343233
pmcid: 9674260
doi: 10.1073/pnas.2207327119
Thakur, A. K. et al. Synthetic multivalent disulfide-constrained peptide agonists potentiate Wnt1/beta-catenin signaling via LRP6 coreceptor clustering. ACS Chem. Biol. 18, 772–784 (2023).
pubmed: 36893429
doi: 10.1021/acschembio.2c00753
Rees, D. C. & Lipscomb, W. N. Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution. Proc. Natl Acad. Sci. USA 77, 4633–4637 (1980).
pubmed: 6933511
pmcid: 349899
doi: 10.1073/pnas.77.8.4633
Rees, D. C. & Lipscomb, W. N. Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 A resolution. J. Mol. Biol. 160, 475–498 (1982).
pubmed: 7154070
doi: 10.1016/0022-2836(82)90309-6
Laps, S., Atamleh, F., Kamnesky, G., Sun, H. & Brik, A. General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins. Nat. Commun. 12, 870 (2021).
pubmed: 33558523
pmcid: 7870662
doi: 10.1038/s41467-021-21209-0
Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 46, D624–D632 (2018).
pubmed: 29145643
doi: 10.1093/nar/gkx1134
Hasan, M. Z., Ikawati, M., Tocharus, J., Kawaichi, M. & Oka, C. Abnormal development of placenta in HtrA1-deficient mice. Dev. Biol. 397, 89–102 (2015).
pubmed: 25446274
doi: 10.1016/j.ydbio.2014.10.015
Li, Y., Salamonsen, L. A., Hyett, J., Costa, F. D. S. & Nie, G. Maternal HtrA3 optimizes placental development to influence offspring birth weight and subsequent white fat gain in adulthood. Sci. Rep. 7, 4627 (2017).
pubmed: 28676687
pmcid: 5496872
doi: 10.1038/s41598-017-04867-3
Liu, J., Li, Y. & Hoh, J. Generation and characterization of mice with a conditional null allele of the HtrA4 gene. Mol. Med. Rep. 12, 6768–6774 (2015).
pubmed: 26353049
pmcid: 4626166
doi: 10.3892/mmr.2015.4291
Mandel, H. et al. Deficiency of HTRA2/Omi is associated with infantile neurodegeneration and 3-methylglutaconic aciduria. J. Med. Genet. 53, 690–696 (2016).
pubmed: 27208207
doi: 10.1136/jmedgenet-2016-103922
Wang Y., Nie G. Overview of human HtrA family proteases and their distinctive physiological roles and unique involvement in diseases, especially cancer and pregnancy complications. Int. J. Mol. Sci. 22, 10756 (2021).
Hara, K. et al. Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N. Engl. J. Med. 360, 1729–1739 (2009).
pubmed: 19387015
doi: 10.1056/NEJMoa0801560
Filliat, G. et al. Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation. PLoS One 12, e0181600 (2017).
pubmed: 28732055
pmcid: 5521800
doi: 10.1371/journal.pone.0181600
Hadfield, K. D. et al. HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains. J. Biol. Chem. 283, 5928–5938 (2008).
pubmed: 18156628
doi: 10.1074/jbc.M709299200
Drummond, E. et al. The amyloid plaque proteome in early onset Alzheimer’s disease and Down syndrome. Acta Neuropathol. Commun. 10, 53 (2022).
pubmed: 35418158
pmcid: 9008934
doi: 10.1186/s40478-022-01356-1
Grau, S. et al. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc. Natl Acad. Sci. USA 102, 6021–6026 (2005).
pubmed: 15855271
pmcid: 1087941
doi: 10.1073/pnas.0501823102
Tennstaedt, A. et al. Human high-temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates. J. Biol. Chem. 287, 20931–20941 (2012).
pubmed: 22535953
pmcid: 3375517
doi: 10.1074/jbc.M111.316232
Baldi, A. et al. The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21, 6684–6688 (2002).
pubmed: 12242667
doi: 10.1038/sj.onc.1205911
Chien, J. et al. A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer. Oncogene 23, 1636–1644 (2004).
pubmed: 14716297
doi: 10.1038/sj.onc.1207271
Lehner, A. et al. Downregulation of serine protease HTRA1 is associated with poor survival in breast cancer. PLoS One 8, e60359 (2013).
pubmed: 23580433
pmcid: 3620283
doi: 10.1371/journal.pone.0060359
Chen, P. H. et al. High-temperature requirement A1 protease as a rate-limiting factor in the development of osteoarthritis. Am. J. Pathol. 189, 1423–1434 (2019).
pubmed: 31051168
doi: 10.1016/j.ajpath.2019.03.013
Grau, S. et al. The role of human HtrA1 in arthritic disease. J. Biol. Chem. 281, 6124–6129 (2006).
pubmed: 16377621
doi: 10.1074/jbc.M500361200
Hou, Y. et al. Lipopolysaccharide increases the incidence of collagen-induced arthritis in mice through induction of protease HTRA-1 expression. Arthritis Rheum. 65, 2835–2846 (2013).
pubmed: 23982886
doi: 10.1002/art.38124
Hu, S. I. et al. Human HtrA, an evolutionarily conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage. J. Biol. Chem. 273, 34406–34412 (1998).
pubmed: 9852107
doi: 10.1074/jbc.273.51.34406
Tsuchiya, A. et al. Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis. Bone 37, 323–336 (2005).
pubmed: 15993670
doi: 10.1016/j.bone.2005.03.015
Wu, J. et al. Comparative proteomic characterization of articular cartilage tissue from normal donors and patients with osteoarthritis. Arthritis Rheum. 56, 3675–3684 (2007).
pubmed: 17968891
doi: 10.1002/art.22876
Ochiai, N. et al. Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment. Commun. Biol. 2, 86 (2019).
pubmed: 30854478
pmcid: 6397181
doi: 10.1038/s42003-019-0334-5
Dewan, A. et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 314, 989–992 (2006).
pubmed: 17053108
doi: 10.1126/science.1133807
Fritsche, L. G. et al. A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat. Genet. 48, 134–143 (2016).
pubmed: 26691988
doi: 10.1038/ng.3448
Yang, Z. et al. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314, 992–993 (2006).
pubmed: 17053109
doi: 10.1126/science.1133811
Agnihotri, S. et al. The genomic landscape of schwannoma. Nat. Genet. 48, 1339–1348 (2016).
pubmed: 27723760
doi: 10.1038/ng.3688
Eigenbrot, C. et al. Structural and functional analysis of HtrA1 and its subdomains. Structure 20, 1040–1050 (2012).
pubmed: 22578544
doi: 10.1016/j.str.2012.03.021
Runyon, S. T. et al. Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3. Protein Sci. 16, 2454–2471 (2007).
pubmed: 17962403
pmcid: 2211686
doi: 10.1110/ps.073049407
Truebestein, L. et al. Substrate-induced remodeling of the active site regulates human HTRA1 activity. Nat. Struct. Mol. Biol. 18, 386–388 (2011).
pubmed: 21297635
doi: 10.1038/nsmb.2013
Skorko-Glonek, J. et al. HtrA protease family as therapeutic targets. Curr. Pharm. Des. 19, 977–1009 (2013).
pubmed: 23016688
doi: 10.2174/1381612811319060003
Zurawa-Janicka, D. et al. Structural insights into the activation mechanisms of human HtrA serine proteases. Arch. Biochem. Biophys. 621, 6–23 (2017).
pubmed: 28396256
doi: 10.1016/j.abb.2017.04.004
Tocharus, J. et al. Developmentally regulated expression of mouse HtrA3 and its role as an inhibitor of TGF-beta signaling. Dev. Growth Differ. 46, 257–274 (2004).
pubmed: 15206957
doi: 10.1111/j.1440-169X.2004.00743.x
Tom, I. et al. Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy. Proc. Natl Acad. Sci. USA 117, 9952–9963 (2020).
pubmed: 32345717
pmcid: 7211935
doi: 10.1073/pnas.1917608117
Schechter, I. & Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–162 (1967).
pubmed: 6035483
doi: 10.1016/S0006-291X(67)80055-X
Ciferri, C. et al. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody. Biochem. J. 472, 169–181 (2015).
pubmed: 26385991
doi: 10.1042/BJ20150601
Gerhardy, S. et al. Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration. Nat. Commun. 13, 5222 (2022).
pubmed: 36064790
pmcid: 9445180
doi: 10.1038/s41467-022-32760-9
Holekamp NMM, L., Brunstein, F., Zhang, J., Wiley, H. & Chen, H. Early termination of a phase 2 study of FHTR2163 in patients with geographic atrophy secondary to age-related macular degeneration. Investig. Ophthalmol. Vis. Sci. 64, 2263 (2023).
Glaza, P. et al. Structural and functional analysis of human HtrA3 protease and its subdomains. PLoS One 10, e0131142 (2015).
pubmed: 26110759
pmcid: 4481513
doi: 10.1371/journal.pone.0131142
Li, W. et al. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat. Struct. Biol. 9, 436–441 (2002).
pubmed: 11967569
doi: 10.1038/nsb795
Acharya, S., Dutta, S. & Bose, K. A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3. Biochem. J. 477, 407–429 (2020).
pubmed: 31899476
doi: 10.1042/BCJ20190706
Chaganti, L. K., Kuppili, R. R. & Bose, K. Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2. FASEB J. 27, 3054–3066 (2013).
pubmed: 23608143
doi: 10.1096/fj.13-227256
Merski, M. et al. Molecular motion regulates the activity of the mitochondrial serine protease HtrA2. Cell Death Dis. 8, e3119 (2017).
pubmed: 29022916
pmcid: 5759095
doi: 10.1038/cddis.2017.487
Singh, N., D’Souza, A., Cholleti, A., Sastry, G. M. & Bose, K. Dual regulatory switch confers tighter control on HtrA2 proteolytic activity. FEBS J. 281, 2456–2470 (2014).
pubmed: 24698088
doi: 10.1111/febs.12799
Lorenzi, T. et al. HtrA1 in human urothelial bladder cancer: a secreted protein and a potential novel biomarker. Int. J. Cancer 133, 2650–2661 (2013).
pubmed: 23712470
Risor, M. W. et al. The autolysis of human HtrA1 is governed by the redox state of its N-terminal domain. Biochemistry 53, 3851–3857 (2014).
pubmed: 24846539
doi: 10.1021/bi401633w
Gandhi, P. S., Chen, Z. & Di Cera, E. Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1. J. Biol. Chem. 285, 15393–15398 (2010).
pubmed: 20236938
pmcid: 2865304
doi: 10.1074/jbc.M110.115337
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
pubmed: 34265844
pmcid: 8371605
doi: 10.1038/s41586-021-03819-2
Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022).
pubmed: 34791371
doi: 10.1093/nar/gkab1061
Martins, L. M. et al. Binding specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi. J. Biol. Chem. 278, 49417–49427 (2003).
pubmed: 14512424
doi: 10.1074/jbc.M308659200
Wenta, T. et al. Cellular substrates and pro-apoptotic function of the human HtrA4 protease. J. Proteom 209, 103505 (2019).
doi: 10.1016/j.jprot.2019.103505
Varallyay, E., Lengyel, Z., Graf, L. & Szilagyi, L. The role of disulfide bond C191-C220 in trypsin and chymotrypsin. Biochem. Biophys. Res. Commun. 230, 592–596 (1997).
pubmed: 9015368
doi: 10.1006/bbrc.1996.6009
Bode, W. & Huber, R. Structural basis of the endoproteinase-protein inhibitor interaction. Biochim. Biophys. Acta 1477, 241–252 (2000).
pubmed: 10708861
doi: 10.1016/S0167-4838(99)00276-9
Radisky, E. S. & Koshland, D. E. Jr. A clogged gutter mechanism for protease inhibitors. Proc. Natl Acad. Sci. USA 99, 10316–10321 (2002).
pubmed: 12142461
pmcid: 124911
doi: 10.1073/pnas.112332899
Laskowski, M. Jr. & Kato, I. Protein inhibitors of proteinases. Annu. Rev. Biochem 49, 593–626 (1980).
pubmed: 6996568
doi: 10.1146/annurev.bi.49.070180.003113
Kocher, S. et al. Tailored Ahp-cyclodepsipeptides as potent non-covalent serine protease inhibitors. Angew. Chem. Int. Ed. Engl. 56, 8555–8558 (2017).
pubmed: 28514117
doi: 10.1002/anie.201701771
Williams, B. L. et al. Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium. Proc. Natl Acad. Sci. USA 118, e2103617118 (2021).
Ganesan, R. et al. Proteolytic activation of pro-macrophage-stimulating protein by hepsin. Mol. Cancer Res. 9, 1175–1186 (2011).
pubmed: 21875933
doi: 10.1158/1541-7786.MCR-11-0004
Lin, S. J., Dong, K. C., Eigenbrot, C., van Lookeren Campagne, M. & Kirchhofer, D. Structures of neutrophil serine protease 4 reveal an unusual mechanism of substrate recognition by a trypsin-fold protease. Structure 22, 1333–1340 (2014).
pubmed: 25156428
doi: 10.1016/j.str.2014.07.008
Shia, S. et al. Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B. J. Mol. Biol. 346, 1335–1349 (2005).
pubmed: 15713485
doi: 10.1016/j.jmb.2004.12.048
Kasperkiewicz, P. et al. Design of a selective substrate and activity based probe for human neutrophil serine protease 4. PLoS One 10, e0132818 (2015).
pubmed: 26172376
pmcid: 4501687
doi: 10.1371/journal.pone.0132818
Zhang, Y. et al. Inhibition of Wnt signaling by Dishevelled PDZ peptides. Nat. Chem. Biol. 5, 217–219 (2009).
pubmed: 19252499
doi: 10.1038/nchembio.152
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
pubmed: 21460441
pmcid: 3069738
doi: 10.1107/S0907444910045749
McCoy, A. J. et al. Phaser crystallographic software. J. Appl Crystallogr. 40, 658–674 (2007).
pubmed: 19461840
pmcid: 2483472
doi: 10.1107/S0021889807021206
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).
pubmed: 31588918
pmcid: 6778852
doi: 10.1107/S2059798319011471
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
pubmed: 20383002
pmcid: 2852313
doi: 10.1107/S0907444910007493
Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).
pubmed: 20124692
pmcid: 2815665
doi: 10.1107/S0907444909047337
UniProt, C. UniProt: the universal protein knowledgebase in 2023. Nucleic Acids Res. 51, D523–D531 (2023).
doi: 10.1093/nar/gkac1052