A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution

Overview

Journal J Med Chem

Publisher American Chemical Society

Specialty Chemistry

Date 2009 Feb 14

PMID 19215089

Citations 28

Authors

Douglas S Auld

Ya-Qin Zhang

Noel T Southall

Ganesha Rai

Marc Landsman

Jennifer MacLure

Daniel Langevin

Craig J Thomas

Christopher P Austin

James Inglese

Affiliations

Soon will be listed here.

Abstract

We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quantitative high-throughput screening (qHTS) was used to determine IC(50)s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC(50) < 10 microM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.

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References

Zavodszky P, Kardos J, Svingor , Petsko G . Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc Natl Acad Sci U S A. 1998; 95(13):7406-11. PMC: 22632. DOI: 10.1073/pnas.95.13.7406. View

Singh P, Harden B, Lillywhite B, Broad P . Identification of kinase inhibitors by an ATP depletion method. Assay Drug Dev Technol. 2004; 2(2):161-9. DOI: 10.1089/154065804323056503. View

Huss K, Blonigen P, Campbell R . Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format. J Biomol Screen. 2007; 12(4):578-84. DOI: 10.1177/1087057107300221. View

Klink T, Kleman-Leyer K, Kopp A, Westermeyer T, Lowery R . Evaluating PI3 kinase isoforms using Transcreener ADP assays. J Biomol Screen. 2008; 13(6):476-85. PMC: 2787408. DOI: 10.1177/1087057108319864. View

Paiardini A, Sali R, Bossa F, Pascarella S . "Hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms. BMC Struct Biol. 2008; 8:14. PMC: 2294123. DOI: 10.1186/1472-6807-8-14. View

Wang W, Xu Q, Shan Y, Xu G . Probing local conformational changes during equilibrium unfolding of firefly luciferase: fluorescence and circular dichroism studies of single tryptophan mutants. Biochem Biophys Res Commun. 2001; 282(1):28-33. DOI: 10.1006/bbrc.2001.4539. View

Cali J, Niles A, Valley M, OBrien M, Riss T, Shultz J . Bioluminescent assays for ADMET. Expert Opin Drug Metab Toxicol. 2008; 4(1):103-20. DOI: 10.1517/17425255.4.1.103. View

Schurer S, Brown S, Gonzalez-Cabrera P, Schaeffer M, Chapman J, Jo E . Ligand-binding pocket shape differences between sphingosine 1-phosphate (S1P) receptors S1P1 and S1P3 determine efficiency of chemical probe identification by ultrahigh-throughput screening. ACS Chem Biol. 2008; 3(8):486-98. PMC: 2597349. DOI: 10.1021/cb800051m. View

Auld D, Southall N, Jadhav A, Johnson R, Diller D, Simeonov A . Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem. 2008; 51(8):2372-86. DOI: 10.1021/jm701302v. View

10.

Schroter T, Minond D, Weiser A, Dao C, Habel J, Spicer T . Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J Biomol Screen. 2008; 13(1):17-28. DOI: 10.1177/1087057107310806. View

11.

Cheng A, Coleman R, Smyth K, Cao Q, Soulard P, Caffrey D . Structure-based maximal affinity model predicts small-molecule druggability. Nat Biotechnol. 2007; 25(1):71-5. DOI: 10.1038/nbt1273. View

12.

Wan P, Garnett M, Roe S, Lee S, Niculescu-Duvaz D, Good V . Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004; 116(6):855-67. DOI: 10.1016/s0092-8674(04)00215-6. View

13.

Hopkins A, Groom C . The druggable genome. Nat Rev Drug Discov. 2002; 1(9):727-30. DOI: 10.1038/nrd892. View

14.

Lowinger T, Riedl B, Dumas J, Smith R . Design and discovery of small molecules targeting raf-1 kinase. Curr Pharm Des. 2002; 8(25):2269-78. DOI: 10.2174/1381612023393125. View

15.

Hajduk P, Greer J . A decade of fragment-based drug design: strategic advances and lessons learned. Nat Rev Drug Discov. 2007; 6(3):211-9. DOI: 10.1038/nrd2220. View

16.

Shewchuk L, Hassell A, Ellis B, Holmes W, Davis R, Horne E . Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail. Structure. 2000; 8(11):1105-13. DOI: 10.1016/s0969-2126(00)00516-5. View

17.

Inglese J, Johnson R, Simeonov A, Xia M, Zheng W, Austin C . High-throughput screening assays for the identification of chemical probes. Nat Chem Biol. 2007; 3(8):466-79. DOI: 10.1038/nchembio.2007.17. View

18.

Fan F, Wood K . Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol. 2007; 5(1):127-36. DOI: 10.1089/adt.2006.053. View

19.

Heitman L, Veldhoven J, Zweemer A, Ye K, Brussee J, IJzerman A . False positives in a reporter gene assay: identification and synthesis of substituted N-pyridin-2-ylbenzamides as competitive inhibitors of firefly luciferase. J Med Chem. 2008; 51(15):4724-9. DOI: 10.1021/jm8004509. View

20.

Manning G, Whyte D, Martinez R, Hunter T, Sudarsanam S . The protein kinase complement of the human genome. Science. 2002; 298(5600):1912-34. DOI: 10.1126/science.1075762. View