Pratistha Ranjitkar

Affinity-Based Probes Based on Type II Kinase Inhibitors

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.

Active site profiling reveals coupling between domains in SRC-family kinases

Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and for performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that display a distinct preference for autoinhibited forms of these kinases. Furthermore, we show that ligands that demonstrate this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.

Reactive Landing of Gas-Phase Ions as a Tool for the Fabrication of Metal Oxide Surfaces for In Situ Phosphopeptide Enrichment

Zirconium, titanium, and hafnium oxide-coated stainless steel surfaces are fabricated by reactive landing of gas-phase ions produced by electrospray ionization of group IVB metal alkoxides. The surfaces are used for in situ enrichment of phosphopeptides before analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To evaluate this method we characterized ZrO2 (zirconia) surfaces by (1) comparison with the other group IVB metal oxides of TiO2 (titania) and HfO2 (hafnia), (2) morphological characterization by SEM image analysis, and (3) dependence of phosphopeptide enrichment on the metal oxide layer thickness. Furthermore, we evaluated the necessity of the reactive landing process for the construction of useful metal oxide surfaces by preparing surfaces by electrospray deposition of Zr, Ti, and Hf alkoxides directly onto polished metal surfaces at atmospheric pressure. Although all three metal oxide surfaces evaluated were capable of phosphopeptide enrichment from complex peptide mixtures, zirconia performed better than hafnia or titania as a result of morphological characteristics illustrated by the SEM analysis. Metal oxide coatings that were fabricated by atmospheric pressure deposition were still capable of in situ phosphopeptide enrichment, although with inferior efficiency and surface durability. We show that zirconia surfaces prepared by reactive landing of gas-phase ions can be a useful tool for high throughput screening of novel phosphorylation sites and quantitation of phosphorylation kinetics.

Affinity Reagents that Target a Specific Inactive Form of Protein Kinases

A number of small-molecule inhibitors have been developed that target the catalytic domains of protein kinases that are not in an active conformation. An inactive form that has been observed in several kinases is the DFG-out conformation. This conformation is characterized by an almost 180 degrees rotation of the conserved Asp-Phe-Gly (DFG) motif in the ATP-binding cleft relative to the active form. However, the sequence and structural determinants that allow a kinase to stably adopt the DFG-out conformation are not known. Here, we characterize a series of inhibitors based on a general pharmacophore for this inactive form. We demonstrate that modified versions of these inhibitors can be used to study the thermodynamics and kinetics of ligand binding to DFG-out-adopting kinases and for enriching these kinases from complex protein mixtures.

Conformation-Selective Inhibitors Reveal Differences in the Activation and Phosphate-Binding Loops of the Tyrosine Kinases Abl and Src

Over the past decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely related tyrosine kinases, such as Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abls activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abls activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases.

Establishing evidence-based thresholds and laboratory practices to reduce inappropriate treatment of pseudohyperkalemia

BACKGROUND Unrecognized pseudohyperkalemia (PHK), defined as an artificial increase in measured potassium concentration, due to thrombocytosis and leukocytosis can lead to inappropriate patient treatment. Understanding the laboratory and patient characteristics that increase risk of PHK is key to preventing diagnostic errors. METHODS Serum/plasma potassium results collected at 2 laboratories over 4years were selected based on blood cell counts collected within 24h and whole blood potassium concentrations determined within 2h of the serum/plasma sample. Differences between whole blood and serum or plasma potassium were compared as functions of platelet or leukocyte count, fit to linear models, and stratified based on leukemia diagnosis codes. Patients having a serum/plasma potassium concentration that was at least 1mEq/mL higher than the whole blood concentration were defined as having PHK. Based on this analysis, high-risk patients were prospectively identified and PHK risk was communicated to providers. Medication administration records were queried to compare rates of kayexalate use pre- and post-intervention. RESULTS Approximately 14% of serum samples with platelet counts >500×109/L had a>1mEq/L increase relative to whole blood potassium. >25% of serum and plasma samples showed a>1mEq/L increase relative to whole blood potassium when leukocyte counts were >50×109/L. Patients with chronic lymphocytic leukemia and high WBC count demonstrated the highest rates of PHK. The rate of kayexalate administration prior to confirmatory testing decreased from 37% to 16% after the laboratory started verbally communicating the possibility of PHK to treating providers. CONCLUSIONS According to our data, a leukocyte count threshold for plasma samples of 50×109/L is appropriate for indicating a high risk of PHK. Direct communication by the laboratory to the care team reduces inappropriate potassium lowering treatment in populations at high risk.

Susceptibility of commonly used ferritin assays to the classic hook effect

*Corresponding author: Dina N. Greene, Department of Laboratory Medicine, University of Washington, Seattle, WA, 98195, USA, Phone: +1 206 5987549, E-mail: dngreene@uw.edu Pratistha Ranjitkar: Department of Laboratory Medicine, University of Washington, Seattle, WA, USA Cameron J. Turtle: Fred Hutchinson Cancer Research Center, Seattle, WA, USA; and Department of Medicine, University of Washington, Seattle, WA, USA Neil S. Harris: Department of Pathology, University of Florida, Gainesville, FL, USA Daniel T. Holmes: Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, BC, Canada; and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Amy Pyle-Eilola: Nationwide Children’s Hospital, Columbus, OH, USA David G. Maloney: Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA; and Seattle Cancer Care Alliance, Seattle, WA, USA Letter to the Editor

Determination of the Kinetics and Thermodynamics of Ligand Binding to a Specific Inactive Conformation in Protein Kinases

Recent interest in inactive kinase conformations has generated the need to develop new biochemical tools to study them. Here, we describe the use of a fluorescent probe that selectively and potently binds to a specific inactive conformation of protein kinases. This allows for the thermodynamics and kinetics of ligand binding to be determined.

Factors influencing naproxen metabolite interference in total bilirubin assays.

BACKGROUND The factors influencing naproxen metabolite O-desmethylnaproxen (ODMN) positive interference in diazo-based Jendrassik and Grof (JG) total bilirubin (Tbil) assays and lack of interference in direct bilirubin (Dbil) assays have not been resolved. The objective of this study was to understand the conditions causing this interference pattern. METHODS Pooled normal and ultra-filtered plasma samples spiked with ODMN and naproxen were measured on the Beckman Coulter DxC and AU instruments. Absorbance spectra were obtained for ODMN mixed with Dbil reagent at original and adjusted pH. Absorbance spectra were also obtained for ODMN and bilirubin samples mixed with Tbil assay reagents. RESULTS ODMN produces a positive interference in the DxC JG Tbil assays, but not the AU Tbil or Dbil assays or the DxC Dbil assay. Neutralizing the acidic pH of AU and DxC Dbil reagents allows ODMN to react with diazo salts. ODMN samples mixed with DxC and AU Tbil reagents produce broad peaks from 450 to 560nm and 400 to 540nm, respectively. The DxC JG Tbil assay monitors a change in absorbance at 520nm close to peak absorbance wavelength of diazo-reacted ODMN, whereas the AU Tbil assay monitors a change in absorbance at 570/660nm, beyond the peak absorbance wavelengths of diazo-reacted ODMN. CONCLUSION The acidic pH of diazo-based Dbil assay reagents inhibits the reaction of ODMN with diazo salts. The AU JG Tbil assay is a reliable method to measure Tbil in the setting of naproxen overdose.

Affinity purification of protein kinases that adopt a specific inactive conformation.

Several protein kinases have been characterized in a specific inactive form called the DFG-out conformation. Unlike the active conformation which is conserved in all kinases, the inactive DFG-out conformation appears to be accessible to only certain kinases. This inactive conformation has been successfully targeted with highly selective kinase inhibitors, including the cancer drugs imatinib and sorafenib. However, the structural and sequence requirements for adopting this conformation are still poorly understood. Here, we describe a general method for enriching DFG-out adopting kinases from cell lysates with an affinity resin that contains a general ligand that specifically recognizes this inactive form.