Enzymatically Cleavable Linker Sequence Motifs for Bioconjugation

Enzyme-cleavable linkers allow connecting two molecules, for example a drug and a carrier, or a probe and a reporter molecule that can be selectively cleaved by specific enzymes. Generally, these linkers are designed to remain stable in normal physiological conditions but are cleaved when exposed to a particular enzyme, triggering the release of the active molecule.

                                  A Summary of Bioconjugates

Enzymes are classified into six different enzyme classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. These hydrolases are widely used to induce chemical-bond cleavage in substrates using water as the nucleophile. Many different types of bonds such as ester, peptide, glycoside, and others can be cleaved but each one requires a specific enzyme such as an esterase, a protease, a glycosidase, or similar. The unique enzyme–substrate specificity distinguishes enzymes from other chemical catalysts and allows the use of enzymes for specific cleavage of peptide substrates or motif. Peptides containing amino acid sequences specific to selected cellular enzymes utilized in enzyme-cleavable linker molecules allow the release of molecular fragments such as drug in cellular compartments.

Enzymatically cleavable linkers are designed to allow targeted release of cargo molecules. The linker is selected such that cleavage occurs only in environments where the target enzyme is present inside specific cells or tissues. For enzyme specificity the linker must contain a sequence or chemical structure selectively recognized and cleaved by a specific enzyme such as a protease, esterase, or glycosidase. Often, peptide linkers are designed to be cleaved by proteases like cathepsin B or matrix metalloproteinases (MMPs). However, ester linkers contain chemical structures that are cleaved by an esterase. Glycosidic linkers are cleaved by glycosidases, and disulfide linkers can be cleaved by redox-sensitive enzymes, for example by thioredoxin.

Applications are selective drug delivery utilizing antibody-drug conjugates (ADCs), diagnostics, imaging, prodrug design, and design of biosensors. In ADCs or Drug Delivery Systems a dipeptide linker like Val-Cit (valine–citrulline) is commonly used which is cleaved by cathepsin B, an enzyme overexpressed in tumor cells. Once cleaved, the active drug is released specifically inside the tumor.

Antibody–Drug Conjugates, or ADCs, allow targeted drug delivery by delivering cytotoxic drugs specifically to diseased or cancer cells and releasing them to the target. Many ADCs contain a cleavable linker that enables controlled drug release in targeted tissues. For example, the Valine–Citrulline (Val–Cit) Linker is cleaved by Cathepsin B, a protease overexpressed in many tumor cells. The Val-Cit linker used in the ADC Brentuximab vedotin (Adcetris®) allows the antibody conjugate to be stable in blood; however, when internalized into lysosomes, the linker is cleaved by Cathepsin B, and the drug is released. The Gly-Phe-Leu-Gly (GFLG) linker is utilized in polymer-drug conjugates for controlled intracellular release and is cleaved by Cathepsin B or other lysosomal proteases. Prodrugs containing ester linkers are cleaved by esterases. In prodrugs, the esterified drug is inactive until it is cleaved in cells.

Fluorescent probes, biosensors, or diagnostics containing enzymatically cleavable linkers release the signal molecule only in the presence of a target enzyme. Fluorescent probes with β-Galactosidase-Cleavable Linkers turn on only in senescent or tumor cells expressing β-Galactosidase. Conjugates with matrix metalloproteinase (MMP)-cleavable peptide linkers are cleaved by MMP-2 and MMP-9. Both MMPs are active in tumor microenvironments, where activatable imaging agents or drug carriers release their payload. Further, enzyme-cleavable linkers allow the designing of materials that degrade or release cargo, for example, growth factors) when specific enzymes are present, for instance, during tissue remodeling or inflammation.

Here is a list of examples of cleavable peptide sequence motifs often used in linkers utilized in applications such as protein purification, imaging enzyme activity and tumor, drug delivery, DNA sequencing, metabolite enrichment.

Enzyme cleavable peptide sequence motifs

Caspase: -DXXD!G(S, A) ; ! = cleavage site

Caspase 1: WEHD!

Caspase 2: VDQQD!

Caspase 3: DEVD!

Caspase 4: LEVD!

Caspase 5: (W/L)EHD!

Caspase 6: (T/V)QVD!

Caspase 7: DEVD!

Caspase 8: LETD!

Caspase 9: LEHD!

Example of a NIR substrate: Cy5.5-GWEHD!GK(FITC)C-NH₂.

{Green DR. Caspases and Their Substrates. Cold Spring Harb Perspect Biol. 2022 Mar 1;14(3):a041012 [PMC]; Guillier F, Orain D, Bradley M. Linkers and cleavage strategies in solid-phase organic synthesis and combinatorial chemistry. Chem Rev. 2000 Jun 14;100(6):2091-158. [PibMed]; Messerli SM, Prabhakar S, Tang Y, Shah K, Cortes ML, Murthy V, Weissleder R, Breakefield XO, Tung CH. A novel method for imaging apoptosis using a caspase-1 near-infrared fluorescent probe. Neoplasia. 2004 Mar-Apr;6(2):95-105. PMC}

Cathepsin B: -R!; -K!F(Y); -R!R-; -FK!-

{Abu Ajaj K, Biniossek ML, Kratz F. Development of protein-binding bifunctional linkers for a new generation of dual-acting prodrugs. Bioconjug Chem. 2009 Feb;20(2):390-6. doi: 10.1021/bc800429q. PMID: 19199576.}

Cathepsin D: --AATYYCQQ!W; -QQWTSN!P; -KAEPKSC!D; -SCDK!T.

Cathepsin D cleaves proteins at specific amino acid residues, typically at hydrophobic positions P1 and P1' (positions relative to the cleavage site), and at P2, P2', P3, and P3'.

{Tung CH, Mahmood U, Bredow S, Weissleder R. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000 Sep 1;60(17):4953-8. PMID: 10987312.; Čaval T, Hecht ES, Tang W, Uy-Gomez M, Nichols A, Kil YJ, Sandoval W, Bern M, Heck AJR. The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle-down characterization of antibodies. FEBS J. 2021 Sep;288(18):5389-5405. doi: 10.1111/febs.15813. Epub 2021 Mar 27. PMID: 33713388; PMCID: PMC8518856. https://pmc.ncbi.nlm.nih.gov/articles/PMC8518856}

Cathepsin K: GHPG!GPQGKC-; -G!K

Cathepsin K, a cysteine protease primarily expressed in osteoclasts, plays a key role in bone resorption. It degrades collagen and other matrix proteins, contributing to the remodeling of bone tissue. One of its motifs is the ERF(W)NIN motif. An example is the CatK peptide substrate GHPG.GPQGKC-NH2 linked to an activatable fluorogenic polymer.

{Jaffer FA, Kim DE, Quinti L, Tung CH, Aikawa E, Pande AN, Kohler RH, Shi GP, Libby P, Weissleder R. Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor. Circulation. 2007 May 1;115(17):2292-8. doi: 10.1161/CIRCULATIONAHA.106.660340. Epub 2007 Apr 9. PMID: 17420353.; Kafienah W, Brömme D, Buttle DJ, Croucher LJ, Hollander AP. Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J. 1998 May 1;331 ( Pt 3)(Pt 3):727-32. doi: 10.1042/bj3310727. PMID: 9560298; PMCID: PMC1219411. https://pmc.ncbi.nlm.nih.gov/articles/PMC1219411/}

Matrix metalloproteinase (MMP): ACPPD; GPAG!LLG; K(Fluorescein)PAG!LLGC-CONH₂, KAG!LLC-CONH₂, and KG!LC-CONH₂; TLPDW!HMNDF

There are three specific collagenases: interstitial collagenase (MMP-1; EC 3.4.24.7), neutrophil collagenase (MMP-8; EC 3.4.24.34) and collagenase-3 (MMP-13; EC 3.4.24.–) (where MMP stands for matrix metalloproteinase). They all initially cleave at a specific Gly-! Leu}Ile bond.

MMPs is a multifunctional enzymes capable of cleaving the extracellular matrix components (collagens, laminin, fibronectin, vitronectin, aggrecan, enactin, versican, perlecan, tenascin, elastin and many others), growth factors, cytokines and cell surface-associated adhesion and signaling receptors.

MMPs including MMP-11, MMP-28 and several MT-MMPs with the furin cleavage motif RXK/RR in their propeptide sequence are normally activated in the trans-Golgi network by serine proteinases such as furin and certain other members of the proprotein convertase family.

{Cieplak P, Strongin AY. Matrix metalloproteinases - From the cleavage data to the prediction tools and beyond. Biochim Biophys Acta Mol Cell Res. 2017 Nov;1864(11 Pt A):1952-1963. doi: 10.1016/j.bbamcr.2017.03.010. Epub 2017 Mar 24. PMID: 28347746; PMCID: PMC5831720. https://pmc.ncbi.nlm.nih.gov/articles/PMC5831720/;

Olson ES, Jiang T, Aguilera TA, Nguyen QT, Ellies LG, Scadeng M, Tsien RY. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4311-6. doi: 10.1073/pnas.0910283107. Epub 2010 Feb 16. PMID: 20160077; PMCID: PMC2840175. https://pmc.ncbi.nlm.nih.gov/articles/PMC2840175/; Tauro JR, Lee BS, Lateef SS, Gemeinhart RA. Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation. Peptides. 2008 Nov;29(11):1965-73. doi: 10.1016/j.peptides.2008.06.021. Epub 2008 Jul 5. PMID: 18652863; PMCID: PMC2592099. https://pmc.ncbi.nlm.nih.gov/articles/PMC2592099/}

TEV protease: ENLYFQ ! G(S); ENLYFQ ! (S/G/A/M/C/H) For recombinant tobacco etch virus (TEV) ptorease, the optimum recognition site for this enzyme is the sequence ENLYFQ(G/S) and cleavage occurs between the Gln and Gly/Ser residues.

{https://en.wikipedia.org/wiki/TEV_protease; Speers AE, Cravatt BF. A tandem orthogonal proteolysis strategy for high-content chemical proteomics. J Am Chem Soc. 2005 Jul 20;127(28):10018-9. PMC}

Thrombin:  RGSPR!XY; RGSPR!FF, RGSPR!S, RGSPR!AA.

{Tanihara M, Suzuki Y, Nishimura Y, Suzuki K, Kakimaru Y. Thrombin-sensitive peptide linkers for biological signal-responsive drug release systems. Peptides. 1998;19(3):421-5. doi: 10.1016/s0196-9781(97)00420-8. PMID: 9533629.}

Trypsin: -aaK ! , -aaR ! Examples: --K!LR!APK!S; -ADYK! DDDDK! -

{Carlson, E. E.; Cravatt, B. F. Chemistry Methods. Nat. Methods 2007, 4, 429; Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9482-7. PMC}

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