Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology.
Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z' factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.
1 Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.|2 State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.
Jing
J
Wang
1 Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.|2 State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.
Pengfei
P
Fang
4 Scripps Research Institute Molecular Screening Center, Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.|PH-Amgen, Inc., Thousand Oaks, CA; PC-Bristol-Myers Squibb, Princeton, NJ.
Peter
P
Chase
5 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Jerusalem 91120, Israel.
Sagi
S
Tshori
5 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Jerusalem 91120, Israel.
Ehud
E
Razin
4 Scripps Research Institute Molecular Screening Center, Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.
Timothy P
TP
Spicer
4 Scripps Research Institute Molecular Screening Center, Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.
Louis
L
Scampavia
4 Scripps Research Institute Molecular Screening Center, Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA.|PH-Amgen, Inc., Thousand Oaks, CA; PC-Bristol-Myers Squibb, Princeton, NJ.
Peter
P
Hodder
1 Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA.|2 State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.|3 Kangma BioTech Ltd., 781 Cailun Road, Shanghai 201203, China.
Min
M
Guo
D000970:Antineoplastic Agents / Q000032:analysis* / Q000494:pharmacology*; D055808:Drug Discovery; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism*; D055503:Protein Multimerization; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
D000970:Antineoplastic Agents; D051739:Microphthalmia-Associated Transcription Factor; D054852:Small Molecule Libraries
AlphaScreen; MITF; high-throughput screening; melanoma