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Site-selective bioconjugation
to protein
Literature seminar
2016/01/23
Takashi Ishiyama

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Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

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What is Bioconjugation?
Bio(生体)conjugation(共役)A : Biomolecules(mainly protein)
B : Molecules adding other function

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Use of Bioconjugation• Creating experimental tool

Immunoassay
Western blotting

Affinity chromatography5/46Use of Bioconjugation• Polyethylene glycol-protein conjugate drugs

Alconcel, S. N. S., Baas, A. S. and Maynard, H. D. Polym. Chem. 2011, 2, 1442

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Use of Bioconjugation• ADC(Antibody-drug conjugate)(Kadcyla)
Trastuzumab(Herceptin)
Inhibition of HER2 signal
emtansine
Inhibition of tubulin polymerization
Stronger therapeutic effect and less side effects
than conventional chemotherapy

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Conditions necessary for bioconjugation• Psysiological conditions not to interfere with proper protein folding and function
Temperature (<37℃)
pH(6-8)
Aqueous solvent• Chemoselectivity(residue-selective reaction)⇒Distinction of different amino acids• Site-selectivity and control of the number of modifications⇒Distinction of local environment of amino acids

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Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

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Importance of control of modification• PEGylation of proteins

Activity of proteins is reduced due to blocking active site by PEG chain
Keefe, A. J. & Jiang, S. Nature Chem. 2012, 4, 59

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Importance of control of modification
Activity of PEGylated chymotrypsin
10090807060504001234501234520
151050

Need for controlled modification to the amino acid away from the active site

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Importance of control of modification• Influence of conjugation site to ADC’s stability and therapeutic activity

Shen, B.-Q. et al. Nature Biotech. 2012, 30, 184• Solvent accessibility : FC-S396C > HC-A114C > LC-V205C• LC-V205C conjugation site is in a positively charged environment

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Importance of control of modification
These ADCs’ in vivo efficacies and pharmacokinetics properties• In vivo activities and durabilities : LC-V205C > HC-A114C >FC-S396C• No differences in total antibody clearance rates⇒Differences in intact ADC levels from linker stability

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Importance of control of modification
Difference of stability of antibody-drug bonding⇒Site-selective bioconjugation is important to improve the stabilityand activity of antibody-drug conjugate

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Importance of control of modification• Influence of conjugation number to ADC’s stability and therapeutic activityHamblett, K. J. et al. Clin. Cancer Res. 2004, 10, 7063

In vitro cytotoxic activities of ADC
IC50…E2 : 6.2 ng/mL
E4 : 2.9 ng/mL
E8 : 1.0 ng/mL

in vitro potency of ADC E2 < E4 < E8

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Importance of control of modification
The effect of drug loading on in vivo antitumor activity

Relationship of ADCs’ activity and DAR or dose
Therapeutic effect
E4 0.5 mg/kg ≓ E8 0.5 mg/kg
E2 1.0 mg/kg > E4 0.5 mg/kg > E8 0.25 mg/kg
in vivo activity isn’t proportional to drug number

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Importance of control of modification
Relationship of conjugation number and pharmacokinetics properties
in vivo stability : E2 > E4 > E8ADCs with higher DAR level are more sensitive
to stress such as temperature.
Beckley, N. S., Lazzareschi, K. P., Chih, H.-W., Sharma, V. K. & Flores, H. L.
Bioconjugate Chem. 2013, 24, 1674.

⇒The number of bioconjugation is also important to improve
the stability and activity of antibody-drug conjugate

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Importance of control of modification• Modification location and number of the protein are importantfor the its activity and stability⇒Site-selective bioconjugation reaction

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Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

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Target of protein bioconjugation• 20 natural amino acids

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Conventional protein bioconjugation
1. Targeting natural lysine
2. Targeting N-terminal amine
3. Targeting natural cysteine21/46

Targeting natural lysine• One of the most nucleophilic functional group in a protein• Abundance on the solvent-exposed outside surfaces of protein⇒Most reactive amino acid

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Targeting natural lysine• Random modification of Lys residues

40 of identified modification sites ⇒ millions of types of products
Modified lysine residues either are located on the protein surface or haverelatively large structural flexibilities.

But site-selectivity is remarkably low
Wang L., Amphlett G., Blattler W.A., Lambert J. M., Zhang W.
Protein Science. 2005. 14. 2436

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Targeting N-terminal amine• Only one N-terminal amine⇒No diversity of conjugation products• Possible to distinct between N-terminal α amine and Lysine ε amine

But impossible to introduce multiple modifications

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Targeting natural cysteine• Rarity of cysteine in proteins⇒Low heterogeneous products• Thiol also has high nucleophilicity• Different reactivity of Thiol (soft nucleophilic group)

But Cys in protein form disulfide bonds⇒Necessity to reduction of S-S bonds

Targeting natural cysteine• Reduction-Alkylation strategies
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Intermolecular and intramolecular disulfide bonds
were distinguished(intermolecular S-S only reduced)
Partial reduction can’t distinguish intermolecular S-Sbonds⇒various reduction product⇒various conjugation productSite-selective Cys reduction-modification was difficult

Sun, M. M. et al. Bioconjugate Chem, 2005, 16, 1282

26/46Strategy of site-selective protein bioconjugation• Impossible to distinguish same amino acids
Lys and Lys, Cys and Cys⇒Recognize other than the target amino acids
Local environment around the reaction point
Ligand recognition site away from reaction point27/46

Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

28/46π-clamp-mediated cysteine bioconjugation• Thiol’s Nucleophilic aromatic substitution (SNAr) reactions

Birchall. J. M. et al. Chem. Comm. 1967. 338• Glutathione-S-transferase are capable of selectivity arylating

Ji. X. et al. Biochemistry. 1993, 32, 12949⇒Arylation of cysteine can be used to Cys-selective bioconjugation.

29/46π-clamp-mediated cysteine bioconjugation• Cys-perfluoroarylation reaction

Cys-selective
Room temperature
But organic solvent needed

Spokoyny. A. M. et al. J. Am. Chem. Com. 2013, 135, 5946

30/46π-clamp-mediated cysteine bioconjugation• Enzyme-catalyzed Cys-bioconjugation
Fast
Cys-serective
Regioselective
Aqueous condition
But enzyme needed

Zhang, C. et al. Angew. Chem. Int. 2013, 52, 14001.

31/46π-clamp-mediated cysteine bioconjugation• Cysteine bioconjugation using without
1. Organic solvent
2. Enzyme (GST)⇒Search of substrates reacting in water⇒Phe-Cys-Pro-Trp-… reacted in water⇒Aromatic amino acids activated the cysteine thiol and interact with theperfluoroaryl group?

32/46π-clamp-mediated cysteine bioconjugation• π-clamp mediated conjugation
Aromatic amino acids accelerated reaction
Only Phe-Cys-Pro-Phe reacted quantitatively
Zhang. C. et al. Nature. Chemistry. 2015, 2413

• Applicable to reaction of protein(antibody)33/46π-clamp-mediated cysteine bioconjugation• Mechanism of π-clamp mediated cysteine bioconjugation
Activation of thiol of Cysteine (stabilization of thiolate)
Structure of Phe-Cys-Pro-Phe←-SH (pKa=7.69±0.09)
More acidic than thiol of Gly-Cys-Pro-Gly (pKa=8.30±0.05)⇒Nucleophilicity of thiol increases

34/46π-clamp-mediated cysteine bioconjugation
Lower activation energy and product free energy
v ΔE = ETS - Epeptide
ΔG = Eproduct + EHF - Epeptide – Eperfluoroaromatics

π-π interaction stabilizes transition state and product⇒reaction acceleration

35/46π-clamp-mediated cysteine bioconjugation
Interaction between reagent and amino acids around target
Cysteine increases site-selectivity of bioconjugation• Problem
Necessity of structure X-Cys-Pro-X to drive bioconjugation reaction⇒Introduction of X-Cys-Pro-X to target protein by genetic manipulation• Next step
Recognition of native amino acids around target residue

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Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

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Ligand-directed selective bioconjugation• Concept of an affinity labeling probe

Site-selective
Protein-selective
Not react to amino acids in active-site

ex.

Chen, G. et al. J. Am. Chem. Soc. 2003, 125, 8130

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Ligand-directed selective bioconjugation• Problem⇒Active site was blocked, reactivity↓⇒Reagents not yield covalent protein-ligand adducts• Ligand-directed Tosyl (LDT) chemistry

T. Tamura, S. Tsukiji and I. Hamachi, J. Am. Chem. Soc., 2012, 134, 2216

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Ligand-directed selective bioconjugation• Design of LDT reagents(spacer structure)40/46

Ligand-directed selective bioconjugation• Reactivity of reagent• Site-selectivity of reagent⇒Spacer D increases reactivity and site-selectivity

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Ligand-directed selective bioconjugation• Consideration of high reactivity and site-selectivity
 Length of spacers b, d is close to the distance
between ligand and target amino acids
 Piperazine type spacer may fix probe towards
Glu57⇒Hard spacer with appropriate length isimportant to reactivity and selectivity

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Ligand-directed selective bioconjugation
Binding of ligand and active site accelerate bioconjugation
reaction• Problem
Slow reaction rate and low labeling efficiency of ligand-directed tocylatechemistry⇒More reactive reagent

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Ligand-directed selective bioconjugation• Ligand-directed acyl imidazole (LDAI) chemistry

Acyl transfer is faster than SN2 reaction?
Much more reactive than LDT reagent
Matsuo. K. et al., Chem. Sci., 2013, 4, 2573
• Ligand-directed dibromophenyl benzoate (LDBB) chemistry
pKa of leaving group : 5.6 (imidazole 14.5)⇒More reactive
Modification yield 50% (30 min)85~90% (3 h)Takaoka. J. et al., Chem. Sci., 2015, 6, 3217

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Ligand-directed selective bioconjugation• Problem
Modification point is limited to around active site
indicated that the original molecular recognition ability of CAII is fully retained.
(S. Tsukiji , et al. J. Am. Chem. Soc., 2009, 131, 9046)⇒Not applicable to modification of amino acids away from active site

• Next step
Recognition of structure of protein surface other than the active site

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Topics
1. Introduction
2. Importance control of bioconjugation
3. Conventional protein modification
4. Site-selective bioconjugation strategy• π-clamp-mediated cysteine bioconjugation• Ligand-directed selective bioconjugation
5. Summary

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SummaryImportance of modification location and number of the protein• Resent study
Transferring the microenvironment of the protein surface to the site-selectivityof bioconjugationπ-clamp-mediated cysteine bioconjugation
Ligand-directed selective bioconjugation• Next step
Development of technique for recognition of protein surfaces