Exploring QPCR-Derived Thermal Melts for Ligand Screening Amanda Jane Meyer Albert Einstein College of Medicine Department of Biochemistry

Thermal Melts by Light Scattering and Fluorescence Stargazer— 384-well format Differential Static Light Scattering Q-PCR— Differential Scanning Fluorescence Sypro Orange Ex:470nM; Em:570nM heat

QPCR Thermal Melt: 9307b1 + MgCl 2

1st derivative of fitted melt-curves

Overview  Biomedically relevant targets: phosphatases and kinases  Community Targets: Functional annotation of the enolase/amidohydrolase superfamilies

protein HOOPO 3 2- ATPADP protein kinase H2OH2O Phosphatases and Kinases H 2 PO 4 - protein phosphatase

Thermal Melting of Pyridoxal Phosphate/Cofilin Phosphatase Using Q-PCR Method PDXP control T m =59.5 T m = ADP-Na, CDP-Na GDP-Na, UDP-Na MgCl 2 T m =65.12 Buffer 100 mM NaCl, 100 mM Glycine/NaOH pH 9.0

Thermal Melting of Pyridoxal Phosphate/Cofilin Phosphatase Using Static Light Scattering 20  L of ~0.4 mg/ml protein/well < 1 mg protein per 100 conditions

control T m =38 0 C PAS Kinase and Screening for Kinase Inhibitors

38 o C PAS Kinase and Screening for Kinase Inhibitors 43 o C 47 o C 49 o C T m -->

Community Targets: Enolases/Amidohydrolases NYSGXRC partnership with NIGMS Evolution of Enzyme Function Program Project Grant – Patsy Babbit (UCSF) - Informatics – John Gerlt, PI (U of Illinois) - Enolases – Frank Raushel (Texas A & M) - Amidohydrolases – Steve Almo (AECOM) – Structural biology – M. Jacobsen, B. Shoichet, A. Sali (UCSF) - Computational Ligand ID Structure-aided functional annotation Structure  Computation  Experimental Validation  Structure

Functional Annotation of an Uncharacterized Thermostable Amidohydrolase From Thermatoga maritima (TM0936) PDB ID: 1P1M R

Amidohydrolase Functional Annotation S-adenosyl-L- homocysteine (SAH) S-inosyl-L- homocysteine (SIH) R Johannes C. Hermann, Ricardo Marti-Arbona, Alexander A. Fedorov, Elena Fedorov, Steven C. Almo, Brian K. Shoichet & Frank M. Raushel Nature 448, (16 August 2007)

TM M GuHCL (T m = ?) TM M GuHCL (T m = 86 o C) Temp. range = 20 – 99 o C (1 o steps) qPCR Thermal Melts of TM0936: Flourescence Based Protein Unfolding Assay Protein = 10  M D

qPCR Thermal Melts of TM0936: Flourescence Based Protein Unfolding Assay D TM M GuHCL (T m = ?) TM M GuHCL (T m = 86 o C) TM M GuHCL + 1mM L-Met (T m = 89 o C) TM M GuHCL + 1mM SIH (T m = 88 o C)

Arginine-specific Carboxypeptidase? 9359b, Amidohydrolase of Unknown Function From Sargasso Sea Environmental Sequencing 3BE7 PDB ID: 3BE7, Patskovsky, Y., Ramagopal, U.A., Toro, R., Meyer, A.J., Freeman, J., Iizuka, M., Bain, K., Rodgers,L., Raushel, F., Sauder, J.M., Burley, S.K., Almo, S.C. (New York Structural GenomiX Research Consortium) Crystal structure of Zn-dependent arginine carboxypeptidase

*[protein] = 1uM (or mg/ml ) 9359b, Amidohydroase From Sargasso Sea Environmental Sequencing

Summary *QPCR-based thermofluor assay can be an effective method for identifying novel ligands for a variety of protein targets. *This method is particularly suited to high-throughput work because large numbers of compounds and/or buffer conditions can be screened simultaneously using a modest amount of protein. *Various classes of proteins (e.g. kinases) can be screened against compound libraries. *QPCR-based thermofluor assay has the potential to improve protein yield, purity, stability in solution, and crystal formation, as well as to provide functional insight

Thank You Structural Genomics Consortium located in Toronto, Canada Special thanks to: Masoud Vedadi Abdellah Allali-Hassani Patrick J. Finerty, Jr. Gregory A. Wasney Irene Chau Guilleimo Senisterra Al Edwards Almo lab members: Rafael Toro JohnJeff Alvarado Jeffrey Bonanno Steve Almo SGX Pharmaceuticals Mike Sauder Shane Atwell Stephen Burley

Thermophilic Proteins Don’t Melt Chaotropes shift the Tm of thermostable proteins. Unfolding transition of 10 uM NYSGXRC target 9272b in the presence of increasing GuHCl concentrations. The unfolding transition occurs at lower temperatures as the GuHCl concentration is increased.

F(T)= F(post) + [F(pre)-F(post)] {1+exp(-  H u /R(1/T-1/T m )+  C pu /R[ln(T/T m )+T m /T-1]} F(T)= fl. Intensity at T T m = midpoint in unfolding transition F(pre)..F(post) = pre/posttransitional fl. Intensities R=gas constant  H u = enthalpy of unfolding  C pu = heat capacity change on protein unfolding In the absence of ligand, T m =T 0,  C pu =  C pu, and  H u =  H u T0T0 T0T0 K L(Tm) = exp{(-  H u /R)/(1/T m -1/T 0 )+(  C pu /R)[ln(T m /T 0 )+T 0 /T m -1]} [L Tm ] K L(Tm) = ligand association constant at T m [L] Tm =free ligand concentration at T m -- ([L] Tm approx = [L] total when [L] total >>[protein] total ) T0T0 T0T0 K L(T) =K L(Tm) * exp{(-  H L(T) /R) * (1/T-1/T m )+(  C PL /R) * [ln(T/T m )+1-(T/T m )]} K L(T) = ligand association constant at temp T  H L(T) = van’t Hoff enthalpy of binding at temp T  C PL = heat capacity change on binding The second exponential term is usually small compared with the first exponential term, so the approximate K L(T) is calculated using…

K L(T) = exp {(-  H L(T) /R) * (1/T-1/T m )}