C-1-06
Screening and Design of Small Molecules that Reversibly
Bind to Streptavidin
Moe Kohno 1
2470493421@mail.ecc.u-tokyo.ac.jp

Takayoshi Okabe 3
tokabe@mol.f.u-tokyo.ac.jp

123

Takuya Terai 1

Shigeru Sugiyama 2t-terai@mol.f.u-tokyo.ac.jp

sugiyama@chem.eng.osaka-u.ac.jp

Tetsuo Nagano 3

Yasuteru Urano 1

tlong@mol.f.u-tokyo.ac.jp

uranokun@m.u-tokyo.ac.jp

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7 -3-1, Hongo,Bunkyo-ku, Tokyo 113-8654, Japan
Graduate School of Science, Osaka University, 1-8, Yamadaoka, Suita-shi,Osaka 565-0871,
Japan
Open Innovation Center for Drug Discovery, The University of Tokyo, 7 -3-1, Hongo,
Bunkyo-ku, Tokyo 113-8654, Japan

Keywords: Ligand docking, Protein-small molecule interaction
The interaction between streptavidin (from Streptmyces avidinii) and biotin is known as thestrongest non-covalent interaction in nature (K d = 10 -13 M) 1 . Streptavidin-biotin system is used formany applications in biotechnology, such as affin ity column chromatography (isolation), SPR
sensor chip (immobilization) or fluorescence imaging (detection). However, for some applications,a few disadvantages of this system have been pointed out. One of the major limitations of thissystem is the almost irreversible binding under physiological conditions. To disrupt a
streptavidin-biotin complex, it is necessary to denature the streptavidin using harsh condition.
In order to make the binding of biotin with streptavidin more easily reversible under mild
conditions, much work has been done to modify streptavidin by genetic mutations. In contrast,
another approach to develop biotin analogs with reduced affinity to streptavidin has been hardlyreported, and the reported biotin analogs provide only partial reversibility. In this research, wescreen a large chemical library to search for novel chemical scaffolds that bind to streptavidin, andaim to optimize the hit compounds based on information from X-ray crystal structure.
First, we performed the screening for avidin (from hen egg) which has identical tertiary structurewith streptavidin. 166,480 compounds were screened by using fluorescence polarization, and weselected 773 compounds as primary hits. These compounds were subsequently screened for
streptavidin, dose-dependently assayed, and analyzed by SPR (Surface Plasmon Resonance). Manybiotin analogs were detected as hits, but we excluded these compounds. Finally, 6 compounds wereselected as novel ligand scaffolds for streptavidin which exhibited fully reversible binding to theprotein (K d = 10 -4 ~10 -6 M). We characterized the binding mode of compound 1 which showed thestrongest affinity (Figure). Based on information from X-ray crystal structure and derivativesynthesis, the amide moiety fixed in cis form was most
likely to be essential for the binding. We synthe size
further derivatives of compound 1 and optimizing theaffinity to streptavidin. Then we will demonstrate theutility of the new reversible scaffold by applying it for
affinity column chromatography.
Blank Subtracted Sensorgrams

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[1] N. M. Green, Adv. Protein Chem., 29:85-133, 1975.

Kd = 3.90×10-6 M
01020[Compound] (μM)

3040200250s