Validation of a Metabolite Panel for a More Accurate Estimation of Glomerular Filtration Rate Using Quantitative LC-MS/MS.

BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.

The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction.

In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) calponin homology (CH)-domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of beta-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t(1/2)) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the calponin homology (CH)-domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess "substrate trapping" properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC(20) in vitro, truncated SMTNL1 lacking the CH-domain was ineffective. In summary, our findings suggest an important role for the CH-domain in mediating the effects of SMTNL1 on smooth muscle contraction.

LC-MS/MS method for quantitation of seven biomarkers in human plasma for the assessment of insulin resistance and impaired glucose tolerance.

Early detection of insulin resistance (IR) and/or impaired glucose tolerance (IGT) is crucial for delaying and preventing the progression toward type 2 diabetes. We recently developed and validated a straightforward metabolite-based test for the assessment of IR and IGT in a single LC-MS/MS method. Plasma samples were diluted with isotopically-labeled internal standards and extracted by simple protein precipitation. The extracts were analyzed by LC-MS/MS for the quantitation of 2-hydroxybutyric acid (0.500-40.0µg/mL), 3-hydroxybutyric acid (1.00-80.0µg/mL), 4-methyl-2-oxopentanoic acid (0.500-20.0µg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50-100µg/mL), oleic acid (10.0-400µg/mL), pantothenic acid (0.0100-0.800µg/mL), and serine (2.50-100µg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1min and the mass spectrometer operated in negative MRM mode. Method validation was performed on three identical LC-MS/MS systems with five runs each. Sufficient linearity (R2>0.99) was observed for all the analytes over the ranges. The imprecision (CVs) was found to be less than 5.5% for intra-run and less than 5.8% for inter-run for the seven analytes. The analytical recovery was determined to be between 96.3 and 103% for the seven analytes. This fast and robust method has subsequently been used for patient sample analysis for the assessment of IR and IGT.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

Growth factor-binding sequence in human alpha2-macroglobulin targets the receptor-binding site in transforming growth factor-beta.

alpha(2)-Macroglobulin (alpha(2)M) binds transforming growth factor-beta1 (TGF-beta1) and TGF-beta2, forcing these growth factors into a state of latency. The mechanism by which this occurs remains unclear. In this paper, we demonstrate that peptides, derived from the structure of human alpha(2)M (amino acids 714-729), bind directly to TGF-beta1 and block the binding of TGF-beta1 to the type I and II TGF-beta receptors. The alpha(2)M-derived peptides are notable for hydrophobic tripeptide sequences (WIW or VVV) and acidic residues (Glu(714) and Asp(719) in the mature alpha(2)M subunit), which may function analogously to the structural elements that mediate TGF-beta-binding in the type II receptor. Mutating Glu(714) and Asp(719) in the alpha(2)M-peptide-GST fusion protein, FP3, which contains the putative growth factor-binding site, significantly decreased the binding affinity of FP3 for TGF-beta1. The alpha(2)M-derived peptides, which bind TGF-beta1, inhibited the interaction of TGF-beta1 with its receptors in fetal bovine heart endothelial cells. The same peptides also inhibited the activity of TGF-beta1 in endothelial cell proliferation assays. These results demonstrate that alpha(2)M-derived peptides target the receptor-binding sequence in TGF-beta.

Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice.

OBJECTIVE: Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. MATERIALS AND METHODS: Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. RESULTS: Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. CONCLUSION: Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.