The University of Electro-communications Taki Laboratory

Artificial molecule-library peptide conjugate on phage:

　We found, for the first time, that cysteine in the library peptide on T7 phage can be selectively modified, while the other cysteines on phage proteins remain intact (Fukunaga, Mol. BioSyst., 2013). Even bachelor students can make the artificial molecule-library peptide conjugate by JUST MIX the library phage and Cysteine (i.e., SH group) reactive reagents, which are widely synthesized and utilized for protein modification. Selected from the artificial library, we can make different 'intelligent targeted' biologics, such as turn-on fluorophores, covalent binders, macrocycles, etc.

Figure. How we found a turn-on fluorophore as the targeted biologics via the 10BASEd-T reaction.

　We have established a novel concept for detection of a target protein by using a keep-on-type fluorescent pharmacophore which was discovered from a dynamic combinatorial library of Schiff bases (Tabuchi, ABC, 2018). As shown in the figure below, the keep-on-pharmacophore exhibits bright fluorescence when irradiated by a UV hand lamp, and the presence of the target protein can be unambiguously detected by naked eye. When the target protein is absent, the keep-on-pharmacophore was degraded by hydrolysis, resulting that we can see no fluorescence.

Figure. Target protein (i.e. HSA)-specific sensing by a keep-on-type fluorescent pharmacophore. HSA was fixed in agarose matrix, incubated with the keep-on-pharmacophore, and UV light of 365 nm was irradiated (dotted-line inset; right panel), and the image was taken by a smartphone.

　N-terminal specific peptide/protein modification via the NEXT-A (N-terminal Extension of protein by Transferase and Aminoacyl-tRNA synthetase) reaction has been invented by us (e.g., Chem. Commun., 2011) . Perhaps, the most useful modification is azide (i.e., N3) introducion to the N-terminus of complicated proteins such as glycosilated antibodies (e.g., Hirasawa, Bioconj. Chem., 2019). This is the fastest enzymatic reaction where 'artificial' substrates can be recognized (Amino Acids, 2015); even radioactive PET probes with short half-life (ca. 1 hour) can be incorporated precisely to the N-terminus of peptide / protein (e.g., CTMC, 2015).

Figure. PET probe introduction via the NEXT-A reaction.

Topic 1: Combinatorially Screened Peptide as Targeted Covalent Binder

Brief summary
　Peptide-type covalent binders for a target protein were obtained by combinatorial screening of nonreactive bait-conjugated peptide libraries on T7 bacteriophage via the 10BASEd-T, followed by structural alteration of the bait into reactive warheads.

Motivation
　Finding targeted covalent binders is one of the cutting-edge disciplines such as biomedical sciences / chemical biology / pharmaceutical fields. Such binders can form permanent bonds to target proteins, and eternally deactivate them. Thus, they would be potentially useful as future medicines (i.e., covalent drugs as antibody-drug substitutes). Here is the first proof-of-concept study for finding peptide-type targeted covalent binder by a combinatorial screening, instead of a rational designing. As shown in the figure, we design a concept for finding peptide covalent binders from solvatochromic bait-modified peptide library[1,2] on T7 bacteriophage constructed via the gp10-based thioetherification (10BASEd-T),[3] followed by structure alteration into reactive warheads[4,5] as bioisosteres.

What has been achieved?
　First, specific introductions of the solvatochromic bait fragments into a designated Cys on displaying library peptides on a capsid protein (i.e., gp10) of T7 bacteriophage were conducted. This 10BASEd-T was carried out without side reactions or loss of phage infectivity.
　Second, from the bait-conjugated peptide library, target-protein (i.e., glutathione S-transferase; GST) specific binders were selected by biopanning; the peptide sequences were analyzed by using DNA sequencers, and consensus sequences for bait-conjugated peptides around the designated Cys were deduced.
　Third, to obtain GST-specific covalent binders, the bait fragments in the consensus peptides were altered into reactive warheads whose structures are shown in Fig. 1.
　Lastly, the chemically-synthesized covalent binders were incubated with GST to facilitate the conjugation.
　In case of using a photo-reactive warhead, ultraviolet (365 nm) irradiation must be needed for the conjugation between the covalent binder and GST; the irradiation simultaneously crosslinks the warhead to the ligand binding site and uncages the fluorescence property of the warhead by forming an intramolecular charge transfer (ICT) structure, which facilitates a rapid confirmation of the successful crosslinking using SDS-PAGE / fluorescence imaging.[5]
　In case of using an always-reactive warhead, such irradiation is unnecessary, and the warhead should theoretically react with limited numbers of amino acids (i.e., nucleophilic ones such as Tyr, Lys, Ser).[4]
　In both cases, site- and position-specific conjugation toward GST was successfully confirmed; the binder-conjugated GST in the SDS-PAGE gel was excised and digested with trypsin, followed by tandem mass spectrometry analysis of the fragment. The specific conjugation was also rationalized by molecular docking simulations of the covalent binders to GST using sievgene of myPresto.[4,5]

References
　1. Taki, M., Inoue, H., Mochizuki, K., Yang, J., and Ito, Y. (2016) Anal. Chem., 88, 1096-1099.
　2. Uematsu, S., Midorikawa, T., Ito, Y., and Taki, M. (2017) AIP Conf. Proc., 1807, 020028.
　3. Fukunaga, K., Hatanaka, T., Ito, Y., Minami, M., and Taki, M. (2014) Chem. Commun., 50, 3921-3923.
　4. Uematsu, S., Tabuchi, Y., Ito, Y., and Taki, M. (2018) Bioconj. Chem., 29, 1866-1871.
　5. Yatabe, K., Hisada, M., Tabuchi, Y., and Taki, M. (2018) Int. J. Mol. Sci., 19, 3682.

Topic 2: Half-life extension of targeted biologics inside of animal bodies

Motivation
　The use of middle molecules (i.e., targeted biologics) in therapeutic applications was limited because of their short half-lifved of these compounds inside bodies, and lack of a suitable bioconjugation method.

What has been achieved?
　We developed a semi-synthetic method of producing Fc-fusion compounds that, unlike conventional recombinant methods, can incorporate artificial middle molecules. The synthesis involves the introduction of an azide group (i.e., N3) to the Fc protein via the N-terminal extension (NEXT-A) reaction developed by our group, and bioconjugation of the middle molecule via strain-promoted azide-alkyne cycloaddition (i.e., SPAAC reaction). The Exenatide-Fc fusion produced through this method not only exhibited a longer plasma half-life, but also retained the biological activity of the original drug through optimization of the length of the spacer between the N-terminus of the protein and the Fc fragment. The method was also successfully applied to a peptide containing non-natural amino acids, a cyclic peptide, and DNA aptamers (Bioconj. Chem., 2019).

Topic 3: Turn-on / keep-on fluorescent molecules as targeted binders

Motivation
　We'd like to establish a general methodology for detection of target proteins by fluorescence-based sensing.

What has been achieved?
　By using an extended phage-display system namely 10BASEd-T [1], peptide-conjugated solvatochromic probes were combinatorially selected (Fig.1A, upper); a conjugation of an optimized peptide to a fluorophore strengthened both specificity and affinity to the target protein along with the solvatochromism, and consequently created a target-specific turn-on sensor [2]. Meanwhile, the fluorophore on the peptide-conjugated probe was altered to a novel photo-crosslinker possessing a caged-fluorescence property (Fig. 1A, lower). The crosslinker worked as a bioisostere of the solvatochromic fluorophore, and eventually, the peptide-conjugated crosslinker bound to the target protein. At this stage, irradiation with UV light simultaneously conjugated the crosslinker with the target at a specific site and uncaged the fluorescence property by forming an intramolecular charge transfer (ICT) structure [3]. Alternatively, as shown in Fig. 1B, a low-molecular-weight pharmacophore, as a targeted fluoroprobe, was also selected from a dynamic combinatorial library of Schiff bases by using size-exclusion chromatography. The identified pharmacophore retained its fluorescence when bound to the hydrophobic site of the target, whereas it lost because of hydrolysis when unbound. We defined it as ?keep-on? type fluorescence probe because the fluorescent pharmacophore is only kept intact when bound to the target [4].

Keywords: targeted biologics, pharmacophore, combinatorial screening, bioisostere.

References
　1. Chem. Commun., 50, (2014) 3921; inside cover article.
　2. Anal. Chem., 88, (2016) 1096; AIP Conf. Proc., 1807, (2017) 020028.
　3. Int. J. Mol. Sci., 19, (2018) 3682.
　4. Anal. Bioanal. Chem., 410, (2018) 6713.