It is an extremely significant and attractive research theme for pharmaceutical study to create new biologically active compounds, clarify life phenomenon process, and further develop therapeutic drugs. I have been working on this theme for more than 40 years relating organic chemistry of amino acids, peptides and proteins to medicinal chemistry.  The outline of my research group is as follows.

 

1. Peptide Chemistry Research

1-1) Development of protection / deprotection methods [1-5]

We found that thioanisole that was regarded as a mere cation scavenger promote the deprotection reaction and established a new concept of deprotection, a push-pull mechanism comprising of a cooperative reaction of a soft nucleophile like thioanisole and a hard electrophile. Currently, deprotection reaction using this method is a standard protocol in peptide synthesis. Further based on this concept, we developed a new deprotection method named Reductive Acidolysis consisting of chlorosilane- trifluoroacetic acid-scavenger systems.

We advanced the Reductive Acidolysis method and enabled the conversion of S-protected cysteine to cystine by reduction in the presence of sulfoxide. By analyzing the reaction mechanism, we developed an originative disulfide forming reaction that is completely different from the conventional method. Based on this method, thus, using stepwise and regioselective crosslinking of three disulfide bridges, we accomplished the total synthesis of human insulin for the first time in the world.

 

1-2) Acid amide forming reactions [6-8]

              We developed in situ neutralization and new uronium-type coupling reagent BOI (Fig. 1) for highly efficient solid phase peptide synthesis. This research led to a new coupling reagent CIP (Fig. 2) that is useful for difficult amino acids. By the study of reaction mechanism, we successfully synthesized an alkaloid, (-)-mirabazole C with consecutive thiazoline scaffold efficiently.

Fig. 1   Fig. 2

 

2. Medicinal Chemistry Research on Peptides

 

2-1) Development of Potent receptor ligands [9-16]

              We performed the structure activity relationship study of an endogenous opioid peptide, enkephalin and human atrial natriuretic peptide (hANP). We found for the first time in the world that a tripeptide analog of enkephalin exhibited analgesic activity by subcutaneous administration. We synthesized C-terminal peptides of hANP that contributed immunochemical research and the result gave a great impact on life science field.

 

2-2) Design and synthesis study of renin inhibitors [17-19]

              Renin is one of aspartic proteases and has high substrate specificity. We paid special attention on the three dimensional interaction between renin and its substrate, and designed and synthesized peptidomimetics containing non-natural amino acid as the substrate transition state mimic.  We developed orally-available renin inhibitor for the first time in the world.

 

2-3) Design and synthesis of HIV protease inhibitors [20-29]

              An aspartic protease, HIV protease, plays important role in proliferation of HIV that is the causative virus of AIDS, and it belongs to the same aspartic protease group as renin.  We used the achievement results of peptide chemistry and medicinal chemistry to challenge the panhuman theme.  We synthesized the HIV protease and its analogs needed for the research with our new and efficient methodologies. Then, we designed and optimized inhibitors based on the substrate transition state concept, and developed highly selective tripeptide inhibitor, KNI-272 (Fig. 3).  The key to success is the unique use of an unnatural amino acid, allophenylnorstatine (Apns), as the substrate recognition site mimic.

 Fig. 3.

 

2-4) Anti-HIV drugs targeting HIV protease active site [25-29]

              We proved experimentally and theoretically that the binding mode of hydroxymethylcarbonyl isostere in KNI-272 to Asp25 and Asp125 in the enzyme active site is same as the substrate transition state. This result is a big and first discovery showing that KNI-272 is the best ideal transition state mimic among many HIV protease inhibitors in the world and that it is a useful tool to reveal enzyme reaction mechanism.

 

2-5) Expansion to medicinal chemistry [30-43]

              We found out that small-sized KNI-272 designed using the world-leading dynamic structural analysis methodology has high inhibitory activity against resistant HIV, oral availability, and good tissue permeability.

              On the other hand, we carried out research on prodrug-type compound aiming at improvement of solubility and membrane-permeability in biological systems. We developed O-N intramolecular acyl migration-type prodrug of HIV protease and improved solubility of the inhibitors.

              We extended these results to spontaneously regenerable and water-soluble prodrug research (Fig. 5) and to O-N intramolecular acyl migration-type water-soluble prodrug of anticancer agent, paclitaxel. 

 

 Fig. 5

 

Furthermore, we are currently expanding difficult sequence peptide synthesis using O-acyl isopeptide method, and synthetic method for Alzheimer’s disease (AD)-related amyloid ß peptide (Aß) (Fig. 6), largely to “Click Peptide” as a new tool for pathological mechanism research for AD.

 Fig. 6

 

2-6) Methodology of protease inhibitor design [44-50]

 

              Emergence of resistant protozoa urges discovery of anti-malarial drugs with new action mechanism. We focused on the aspartic protease, plasmepsin, which is specific and important for nutrition intake in the protozoa and developed new anti-malarial compounds based on the substrate-transition state concept accumulated during renin and HIV protease inhibitor research.  These compounds were shown to suppress proliferation of malarial protozoa in red blood cells. Furthermore, he designed and synthesized highly potent plasmepsin inhibitor KNI-10006 (Fig. 7) using protein space search methodology.

 Fig. 7

 

              We applied thus established methodology of aspartic protease inhibitor design to the inhibitor research of ß-secretase (BACE1) which modulates the formation of Aß. We achieved synthesis of highly potent inhibitors, their size reduction and chemical stabilization. The low molecular-weight BACE1 inhibitor thus obtained, KMI-429, exhibited high potency in cell assay system, and in vivo Aß production inhibition for the first time in the world.  This result attracts worldwide attention as the basic research for AD therapeutics discovery.

 

              The first paper of KNI-272 was published in Japanese Pharmaceutical Society Journal written in English and attracted much attention with 115 citations.  The photograph of the HIV protease-KNI-272 complex revealed by x-ray crystallographic analysis was shown in a review published in Nature Reviews Drug Discovery and adopted as the front cover picture of some journals. These facts show the huge impact of our research.

              Particularly, KNI-10006 was highlighted as “Searching Adaptive Space” in Science, which shows high evaluation of Apns scaffold concept.  Water-soluble prodrug research was introduced in “Editor’s Eye” column in a journal published by Japanese Pharmaceutical Society, and the high usefulness of the prodrug is immensely evaluated there.  In addition, KMI-429 was introduced in major newspapers and magazines describing that it gave a great impact in AD therapeutic research.

 

 

Representative Publications

1. Y. Kiso, K. Ukawa, T. Akita, Efficient removal of N-benzyloxycarbonyl group by a 'push-pull' mechanism using thioanisole-trifluoroacetic acid, exemplified by a synthesis of Met-enkephalin. J. Chem. Soc., Chem. Commun., 101-102 (1980).

2. Y. Kiso, T. Kimura, M. Yoshida, M. Shimokura, K. Akaji, T. Mimoto, A new class of amino protecting group removable by reductive acidolysis: 4-methylsulphinyl-benzyloxycarbonyl (Msz) group. J. Chem. Soc., Chem. Commun., 1511-1513 (1989).

3. Y. Kiso, T. Kimura, Y. Fujiwara, H. Sakikawa, K. Akaji, Efficient solid phase peptide synthesis on a phenacyl-resin by a methanesulfonic acid a-amino deprotecting procedure. Chem. Pharm. Bull. 38, 270-272 (1990).

4. K. Akaji, T. Tatsumi, M. Yoshida, T. Kimura, Y. Fujiwara, Y. Kiso, Disulfide bond formation using the silyl chloride-sulfoxide system for the synthesis of a cystine peptide. J. Am. Chem. Soc., 114, 4137-4143 (1992).

5. K. Akaji, K. Fujino, T. Tatsumi, Y. Kiso. Total synthesis of human insulin by regioselective disulfide formation using the silyl chloride-sulfoxide.J. Am. Chem. Soc. 115, 11384-11392 (1993).

6. Y. Kiso, Y. Fujiwara, T. Kimura, A. Nishitani, K. Akaji, Efficient solid phase peptide synthesis: use of methanesulfonic acid α-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethyl imidazolidiniumhexafluorophosphate (BOI). Int. J. Peptide & Protein Res., 40, 308-314 (1992).

7. K. Akaji, N. Kuriyama, Y. Kiso, Efficient coupling of a,a-dimethyl amino acid using a new chloroimidazolidium reagent, CIP. Tetrahedron Letters, 35, 3315-3318 (1994).

8. K. Akaji, N. Kuriyama, Y. Kiso. Convergent synthesis of (-)-mirabazole C using a chloroimidazolidium coupling reagent, CIP. J. Org. Chem. 61, 3350-3357 (1996).

9. Y. Kiso, M. Yamaguchi, T. Akita, H. Moritoki, M. Takei, H. Nakamura, Super-active enkephalin analogues. Simple tripeptide hydroxyalkylamide exhibit surprisingly high and long-lasting opioid activities. Naturwissenschaften, 68, 210-212 (1981).

10. K. Nakao, A. Sugawara, N. Morii, M. Sakamoto, M. Suda, J. Soneda, T. Ban, M. Kihara, Y. Yamori, M. Shimokura, Y. Kiso, H. Imura, Radioimmunoassay for a-human and rat atrial natriuretic polypeptide. Biochem. Biophys. Res. Commun., 124, 815-821 (1984).

11. N. Morii, K. Nakao, A. Sugawara, M. Sakamoto, M. Suda, M. Shimokura, Y. Kiso, M. Kihara, Y. Yamori, H. Imura, Occurrence of atrial natriuretic polypeptide in brain. Biochem. Biophys. Res. Commun., 127, 413-419 (1985).

12. A. Sugawara, K. Nakao, N. Morii, M. Sakamoto, M. Suda, M. Shimokura, Y. Kiso, M. Kihara, Y. Yamori, K. Nishimura, J. Soneda, T. Ban, H. Imura, a-Human atrial natriuretic polypeptide is released from the heart and circulates in the body. Biochem. Biophys. Res. Commun., 129, 439-446 (1985).

13. M. Kawata, K. Nakao, N. Morii, Y. Kiso, H. Yamashita, H. Imura, Y. Sano, Atrial natriuretic polypeptide: Topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry. Neuroscience, 16, 521-546 (1985).

14. Y. Kiso, M. Shimokura, S. Hosoi, T. Fujisaki, Y. Fujiwara, M. Yoshida, Syntheses and biological activities of atrial natriuretic polypeptide analogs. J. Protein Chem., 6, 147-162 (1987).

15. M. Hashimoto, K. Takada, Y. Kiso, S. Muranishi, Synthesis of palmitoyl derivatives of insulin and their biological activities. Pharmaceut .Res. 6, 171-176 (1989).

16. T. W. von Geldern, G. P. Budzik, T. P. Dillon, W. H. Holleman, M. A. Holst, Y. Kiso, E. I. Novosad, T. J. Opgenorth, T. W. Rockway, A. M. Thomas, S. Yeh. Atrial natriuertic peptide antagonists: Biological evaluation and structural correlations. Mol. Pharmacol., 38, 771-778 (1990).

17. K. Iizuka, T. Kamijo, T. Kubota, K. Akahane, H. Umeyama, Y. Kiso, New human renin inhibitors containing an unnatural amino acid norstatine. J. Med. Chem., 31, 701-704 (1988).

18. K. Iizuka, T. Kamijo, H. Harada, K. Akahane, T. Kubota, H. Umeyama, Y. Kiso, Design and synthesis of an orally potent human renin inhibitor containing a novel amino acid, cyclohexylnorstatine. J. Chem. Soc. Chem. Commun., 1678-1680 (1989).

19. K. Iizuka, T. Kamijo, H. Harada, K. Akahane, T. Kubota, H. Umeyama, T. Ishida, Y. Kiso. Orally potent human renin inhibitors derived from angiotensinogen transition state: design, synthesis, and mode of interaction. J. Med. Chem., 33, 2707-2714 (1990).

20. T. Mimoto, J. Imai, S. Tanaka, N. Hattori, O. Takahashi, S. Kisanuki, Y. Nagano, M. Shintani, H. Hayashi, H. Sakikawa, K. Akaji, Y. Kiso, Rational design and synthesis of a novel class of active site-targeted HIV protease inhibitors containing a hydroxymethylcarbonyl isostere. Use of phenylnorstatine or allophenylnorstatine as a transition-state mimic.  Chem. Pharm. Bull., 39, 2465-2467 (1991).

21. T. Mimoto, J. Imai, S. Tanaka, N. Hattori, S. Kisanuki, K. Akaji, Y. Kiso,KNI-102, a novel tripeptide HIV protease inhibitor containing allophenylnorstatine as a transition-state mimic.  Chem. Pharm. Bull., 39, 3088-3090 (1991).

22. T. Mimoto, J. Imai, S. Kisanuki, H. Enomoto, N. Hattori, K. Akaji, Y. Kiso, Kynostatin (KNI)-227 and -272, highly potent anti-HIV agents: Conformationally constrained tripeptide inhibitors of HIV protease containing allophenylnorstatine.Chem. Pharm. Bull., 40, 2251-2253 (1992).

23. S. Kageyama, T. Mimoto, Y. Murakawa, M. Nomizu, H. Ford, Jr., T. Shirasaka, S. Gulnik, J. Erickson, K. Takada, H. Hayashi, S. Broder, Y. Kiso, H. Mitsuya. In vitro anti-HIV activity of transition-state mimetic HIV protease inhibitors containing allophenylnorstatine. Antimicrob. Agent Chemother., 37, 810-817 (1993).

24. Y. Kiso, Design and synthesis of substrate-based peptidomimetic HIV protease inhibitors containing the hydroxymethylcarbonyl isostere. Biopolymers 40, 235-244 (1996).

25. E. T. Baldwin, T. N. Bhat, S. Gulnik, B. Liu, I. A. Topol, Y. Kiso, T. Mimoto, H. Mitsuya, J. W. Erickson. Structure of HIV-1 protease with KNI-272, a tight-binding transition-state analog containing allophenylnorstatine.Structure, 3, 581-590 (1995).

26. Y.-X. Wang, D. I. Freedberg, T. Yamazaki, P. T. Wingfield, S. J. Stahl, J. D. Kaufman, Y. Kiso, D. A. Torchia, Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272. Biochemistry, 35, 9945-9950 (1996).

27. Y.-X. Wang, D. I. Freedberg, P. T. Wingfield, S. J. Stahl, J. D. Kaufman, Y. Kiso, T. N. Bhat, J. W. Erickson, D. A. Torchia: Bound water molecules at the interface between the HIV-1 protease and a potent inhibitor, KNI-272, determined by NMR. J. Am. Chem. Soc., 118, 12287-12290 (1996).

28. D. I. Freedberg, Y.-X. Wang, S. J. Stahl, J. D. Kaufman, P. T. Wingfield, Y. Kiso, D. A. Torchia.  Flexibility and function in HIV protease: Dynamics of the HIV-1 protease bound to the asymmetric inhibitor kynostatin 272 (KNI-272).  J. Am. Chem. Soc., 120, 7916-7923 (1998).

29. E. Katoh, T. Yamazaki, Y. Kiso, P. T. Wingfield, S. J. Stahl, J. D. Kaufman, D. A. Torchia. Determination of the rate of monomer interchange in a ligand-bound homodimeric protein from NOESY cross peaks: application to the HIV protease/KNI-529 complex. J. Amer. Chem. Soc., 121, 2607-2608 (1999).

30. S. Kageyama, B. D. Anderson, B. L. Hoesterery, H. Hayashi, Y. Kiso, K. P. Flora, H. Mitsuya, Protein binding of human immunodeficiency virus protease inhibitor KNI-272 and alteration of its in vitro antiretroviral activity in the presence of high concentrations of proteins. Antimicrob. Agents Chemother., 38, 1107-1111 (1994).

31. T. Mimoto, R. Kato, H. Takaku, S. Nojima, K. Terashima, S. Misawa, T. Fukazawa, T. Ueno, H. Sato, M. Shintani, Y. Kiso, H. Hayashi. Structure-activity relationship of small-sized HIV protease inhibitors containing allophenylnorstatine.J. Med. Chem., 42, 1789-1802 (1999).

32. Y. Kiso, H. Matsumoto, S. Mizumoto, T. Kimura, Y. Fujiwara, K. Akaji. Small dipeptide-based HIV protease inhibitors containing the hydroxymethylcarbonyl isostere as an ideal transition-state mimic. Biopolymers, 51, 59-68 (1999).

33. T. Mimoto, N. Hattori, H. Takaku, S. Kisanuki, T. Fukazawa, K. Terashima, R. Kato, S. Nojima, S. Misawa, T. Ueno, J. Imai, H. Enomoto, S. Tanaka, H. Sakikawa, M. Shintani, H. Hayashi, Y. Kiso. Structure-activity relationship of orally potent tripeptide-based HIV proteae inhibitors containing hydroxymethylcarbonyl isostere.Chem. Pharm. Bull., 48, 1310-1326 (2000).

34. H. Matsumoto, T. Kimura, T. Hamawaki, A. Kumagai, T. Goto, K. Sano, Y. Hayashi, Y. Kiso. Design, synthesis, and biological evaluation of anti-HIV double-drugs: conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor through spontaneously cleavable linkers. Bioorg .Med. Chem., 9, 1589-1600 (2001).

35. Y. Hamada, J. Ohtake, Y. Sohma, T. Kimrua, Y. Hayashi, Y. Kiso. New water-soluble prodrugs of HIV protease inhibitors based on OｮN intramolecular acyl migration. Bioorg .Med. Chem., 10, 4155-4167 (2002).

36. Y. Sohma, Y. Hayashi, T. Ito, H. Matsumoto, T. Kimura, Y. Kiso: Development of water-soluble prodrugs of the HIV-1 protease inhibitor KNI-727: Importance of the conversion time for higher gastrointestinal absorption of prodrugs based on spontaneous chemical cleavage. J. Med. Chem. 46, 4124-4135 (2003).

37. Y. Hayashi, M. Skwarczynski, Y. Hamada, Y. Sohma, T. Kimura, Y. Kiso: A novel approach of water-soluble paclitaxel prodrug with no auxiliary and no byproduct: Design and synthesis of isotaxel. J. Med. Chem., 46, 3782-3784 (2003).

38. Y. Sohma, M. Sasaki, Y. Hayashi, T. Kimura, Y. Kiso: Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides. Chem. Commun., 124-125 (2004).

39. Y. Sohma, M. Sasaki, Y. Hayashi, T. Kimura, Y. Kiso: Design and synthesis of a novel water-soluble Aß 1-42 isopeptide: an efficient strategy for the preparation of Alzheimers disease-related peptide, Aß 1-42, via O-N intramolecular acyl migration reaction. Tetrahedron Letters, 45, 5965-5968 (2004).

40. Y. Sohma, Y. Hayshi, M. Skwarczynski, Y. Hamada, M. Sasaki, T. Kimura, Y. Kiso: O-N Intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides. Biopolymers, 76, 344-356 (2004).

41. M. Skwarczynski, Y. Sohma, M. Noguchi, M. Kimura, Y. Hayashi, Y. Hamada, T. Kimura, Y. Kiso: No auxiliary, no byproduct strategy for water-soluble prodrugs of taxoids: scope and limitation of O-N intramolelcular acyl and acyloxy migration reaction. J. Med. Chem. 48, 2655-2666 (2005).

42. A. Taniguchi, Y. Sohma, M. Kimura, T. Okada, K. Ikeda, Y. Hayashi, T. Kimura, S. Hirota, K. Matsuzaki, Y. Kiso: ‘Click peptide’ based on the ‘O- aclisopeptide method’: control of A ß 1-42 production from a photo-triggered A ß 1-42 analogue. J. Am. Chem. Soc., 128, 696-697 (2006).

43. Y. Sohma, Y. Kiso, “Click peptides”—chemical biology-oriented synthesis of Alzheimer’s disease-related amyloid ß peptide (Aß) analogues based on the ‘O-acyl isopeptide method’. ChemBioChem, published online on 17 Aug (2006).

44. A. Nezami, I. Luque, T. Kimura, Y. Kiso, E. Freire: Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target. Biochemistry, 41, 2273-2280 (2002).

45. A. Nezami, T. Kimura, K. Hidaka, A. Kiso, J. Liu, Y. Kiso, D. E. Goldberg, E. Freire: High affinity inhibition of a family of Plasmodium falciparumproteaes by a designed adaptive inhibitor. Biochemistry, 42, 8459-8464 (2003).

46. D. Shuto, S. Kasai, T. Kimura, P. Liu, K. Hidaka, T. Hamada, S. Shibakawa, Y. Hayashi, C. Hattori, B. Szabo, S. Ishiura, Y. Kiso: KMI-008, a novel ß-secretase inhibitor containing a hydroxymethylcarbonyl isostere as a transition-state mimic: design and synthesis of substrate-based octapeptides. Bioorg .Med. Chem. Lett., 13, 4273-4276 (2003).

47. T. Kimura, D. Shuto, S. Kasai, P. Liu, K. Hidaka, T. Hamada, Y. Hayashi, C. Hattori, M. Asai, S. Kitazume, T. C. Saido, S. Ishiura, Y. Kiso. KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine. Bioorg .Med. Chem. Lett., 14, 1527-1531 (2004).

48. T. Kimura, D. Shuto Y. Hamada, N. Igawa, S. Kasai, P. Liu, K. Hidaka, T. Hamada, Y. Hayashi, Y. Kiso: Design and synthesis of highly active Alzheimer’s ß-secretase (BACE1) inhibitors, KMI-420 and KMI-429, with enhanced chemical stability. Bioorg. Med. Chem. Lett., 15, 211-215 (2005).

49. M. Asai, C. Hattori, N. Iwata, T. C. Saido, N. Sasagawa, B. Szabo, Y. Hasimoto, K. Maruyama, S. Tamura, Y. Kiso, S. Ishiura: The novel ß-secretase inhibitor KMI-429 reduces amyloid ß peptide production in amyloid precursor protein transgenic and wild-type mice J. Neurochem., 96, 533-540 (2006).

50. Z. Ziora, T. Kimura, Y. Kiso, Small-sized BACE1 inhibitors. Drugs of the Future, 31, 53-63 (2006).