Monitoring anti-angiogenic therapy in colorectal cancer murine model using dynamic contrast-enhanced MRI: comparing pixel-by-pixel with region of interest analysis.

Sorafenib is a multi-kinase inhibitor that blocks cell proliferation and angiogenesis. It is currently approved for advanced hepatocellular and renal cell carcinomas in humans, where its major mechanism of action is thought to be through inhibition of vascular endothelial growth factor and platelet-derived growth factor receptors. The purpose of this study was to determine whether pixel-by-pixel analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is better able to capture the heterogeneous response of Sorafenib in a murine model of colorectal tumor xenografts (as compared with region of interest analysis). MRI was performed on a 9.4 T pre-clinical scanner on the initial treatment day. Then either vehicle or drug were gavaged daily (3 days) up to the final image. Four days later, the mice were again imaged. The two-compartment model and reference tissue method of DCE-MRI were used to analyze the data. The results demonstrated that the contrast agent distribution rate constant (K(trans)) were significantly reduced (p < 0.005) at day-4 of Sorafenib treatment. In addition, the K(trans) of nearby muscle was also reduced after Sorafenib treatment. The pixel-by-pixel analysis (compared to region of interest analysis) was better able to capture the heterogeneity of the tumor and the decrease in K(trans) four days after treatment. For both methods, the volume of the extravascular extracellular space did not change significantly after treatment. These results confirm that parameters such as K(trans), could provide a non-invasive biomarker to assess the response to anti-angiogenic therapies such as Sorafenib, but that the heterogeneity of response across a tumor requires a more detailed analysis than has typically been undertaken.

Quantitative analysis of water proton spectral lineshape: a novel source of contrast in MRI.

Previous work in this laboratory has demonstrated improved anatomic and functional images produced from high spectral and spatial resolution (HiSS) MRI of the water proton signal. The present work tests the hypothesis that different Fourier components of the water resonance represent anatomically and/or physiologically distinct populations of water molecules within each small image voxel. HiSS datasets were acquired from tomatoes and rodent tumors at 4.7 T using echo-planar spectroscopic imaging (spatial and spectral resolutions were 117-150 microm and 1.5-3.1 Hz, respectively). Images of each Fourier component of the water resonance (referred to as Fourier component images, or FCIs) were produced. FCIs at frequencies offset from the peak of the water resonance ('off-peak' FCIs) were compared to images of the Fourier component with largest amplitude, i.e. the water peak-height image. Results demonstrate that off-peak FCIs differ significantly from the water peak-height image and that water resonances are often asymmetric. These results show that water signal at various frequency offsets from the peak of the water resonance come from water molecules in different anatomic/physiologic environments. Off-peak FCIs are a new source of structural and functional information and may have clinical utility.

ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst.

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.

Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods.

An expression system has been developed that allows high levels of production of TEM-1 beta-lactamase with ease of biosynthetic incorporation of nuclear isotopes. The gene for mature TEM-1 beta-lactamase fused to the leader sequence of the ompA protein was subcloned into the pET-24a(+) vector by introduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induction at 25 degrees C supported by LB medium supplemented with osmolytes (300 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-type TEM-1 beta-lactamase was recovered at a level of 140 mg/L. The production level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the efficiency of biosynthetic incorporation of covalent deuterium showed maximal (90%) incorporation with minimal medium containing 99% (2)H(2)O and sodium d(3)-acetate (99 atom% (2)H). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced. The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labeled penicillin substrate and hydrogens on the penicillin moiety in a cryokinetically isolated acylenzyme reaction intermediate because of the decrease in overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies requiring large quantities of protein in isotopically enriched forms.

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

ENDOR studies of VO2+: probing protein-metal ion interactions in nephrocalcin.

Nephrocalcin inhibits the growth of calcium oxalate monohydrate crystals in the mammalian kidney. Isoforms A and B contain three equivalents of gamma-carboxyglutamic acid (Gla) residues implicated in Ca2+-binding and exhibit strong inhibitor properties and high Ca2+-binding affinity (Kd approximately 10(-8) M). Isoforms C and D lack these properties and exhibit low Ca2+-binding affinity (Kd approximately 10(-6) M). With VO2+ as a structural probe, electron paramagnetic resonance (EPR) studies of the Ca2+-binding sites of isoforms B and D showed that VO2+ binds competitively with a metal ion:protein stoichiometry of 4:1. EPR spectral parameters of the VO2+ ion were consistent with only equatorial oxygen-donor ligands. EPR and angle-selected electron nuclear double resonance (ENDOR) spectra showed two equatorially positioned, metal coordinating waters in isoform D while in isoform B no ligands undergoing hydrogen exchange were found. Since isoform D showed no evidence for axially coordinated water, similarly to isoform B, it is likely that the protein residues occupying the axial sites are identical in both proteins. ENDOR spectra of VO2+-complexes of isoforms B and D were compared to spectra of the VO2+-complex with alpha-ethylmalonic acid (EMA), a molecular mimic of Gla. Spectra of the VO2+-complex of EMA showed axial water located trans to the V=O bond and outer shell water hydrogen-bonded to the vanadyl oxygen, consistent with the X-ray structure of Ca(EMA)2. We, therefore, conclude that the spatial disposition of carboxylate groups of Gla residues coordinating Ca2+ in isoforms A and B must differ from that observed in the crystal structure of Ca(EMA)2.

Characterization of Ca(2+)-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin using vanadyl ions: different metal binding properties in strong and weak inhibitor proteins revealed by EPR and ENDOR.

Nephrocalcin (NC), a calcium-binding glycoprotein of 14,000 molecular weight as a monomer, is known to inhibit the growth of calcium oxalate monohydrate (COM) crystals in renal tubules. We have isolated NC from bovine kidney tissue and purified into four isoforms, fractions A-D. NC-A and NC-B strongly inhibit the growth of COM crystals, and NC-C and NC-D inhibit crystal growth weakly. The strongly inhibitor proteins are abundant in normal subjects, whereas stone formers excrete less of NC-A and NC-B and more of NC-C and NC-D. NC-C was characterized with respect to its metal binding sites by using vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. We demonstrated that VO2+ binds to NC-C with a stoichiometry of metal:protein binding of 4:1 and that VO2+ competes with Ca2+ in binding to NC-C. In NC-C, the metal ion is exposed to solvent water molecules and two water molecules are detected in the inner coordination sphere of the metal ion by ENDOR. In the metal binding environment of NC-A, as reported previously (Mustafi, D., & Nakagawa, Y. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11323-11327), inner sphere coordinated water is completely excluded. Based on the results of the metal binding properties in both strong and weak inhibitor proteins, a probable mechanism of inhibition of COM crystal growth by NC has been outlined.

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.

Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

The structure of the acylenzyme intermediate in the hydrolysis of the specific spin-label ester substrate methyl N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-tryptophan ate and its fluoro analogs catalyzed by alpha-chymotrypsin (EC 3.4.21.1) has been determined by electron nuclear double resonance (ENDOR) and molecular modeling methods. By a combination of kinetic and cryoenzymological methods, we have established conditions to stabilize the spin-labeled acylenzyme reaction intermediate. Proton ENDOR features specific for the substrate were assigned on the basis of specific deuteration. From the observed ENDOR shifts for protons and fluorines that correspond to principal hyperfine coupling components, the dipolar hyperfine coupling contributions were calculated to estimate electron-nucleus distances. With these dipolar separations as constraints, conformations of the substrate both free in solution and in the active site of alpha-chymotrypsin were determined by molecular graphics analysis. Comparison of the conformation of the bound substrate to that of the free substrate showed that formation of the acylenzyme requires significant torsional alteration in substrate structure. The structural relationships between active-site residues and the substrate in its ENDOR-assigned conformation are examined with respect to the requirements of stereoelectronic rules for formation and breakdown of the acylenzyme species.

Catalytic conformation of carboxypeptidase A. Structure of a true enzyme reaction intermediate determined by electron nuclear double resonance.

The structure of a catalytically competent reaction intermediate of carboxypeptidase A (CPA) formed with the specific spin-label ester substrate O-[3-(2,2,-5,5-tetramethyl-1-oxypyrrolinyl)propen-2-oyl]-L-b eta- phenyllactate through application of cryoenzymological methods has been determined by electron nuclear double resonance (ENDOR) and molecular modeling. It is shown that the reaction intermediate is best identified as a mixed-anhydride acylenzyme derivative in which the side chain of Glu-270 is acylated by the spin-label substrate, in agreement with previous cryoenzymological and spectroscopic studies from this laboratory. From the observed proton ENDOR shifts corresponding to principal hyperfine coupling components and assigned by selective deuteration, the dipolar hyperfine coupling components were calculated to estimate electron-proton distances. With these ENDOR-determined distances as constraints, the conformation of the substrate free in solution and in the active site of CPA has been determined on the basis of torsion angle search calculations. With a catalytically active, acetylated form of CPA, we have also assigned the position of the side chain of Tyr-198 with respect to the nitroxyl group. The positional assignments of both substrate and active-site residues in a true reaction intermediate provide important constraints in defining the structural basis of action of CPA.

Structure and conformation of the nitroxyl spin-label ethyl 3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oate determined by electron nuclear double resonance: comparison with the structure of a spin-label substrate of carboxypeptidase A.

The conformation of the nitroxyl spin-label ethyl 3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oate has been determined by electron nuclear double resonance (ENDOR) spectroscopy and computer-based molecular modeling. From ENDOR spectra of the compound in frozen solution, we have assigned resonance absorption features for each class of protons, and we have identified their principal hyperfine coupling (hfc) components from analysis of the dependence of ENDOR spectra on the static laboratory magnetic field. The dipolar hfc components yielded estimates of the electron-proton separations for each class of protons of the ethyl propenoyl moiety. Torsion angle search calculations were carried out to determine the conformational space compatible with hard-sphere nonbonded constraints and with the ENDOR-determined distance constraints. Molecular graphics analysis revealed that the propenoyl side chain of the spin-label exhibits an extended trans conformation and that the ethyl moiety of the ester group deviates significantly from coplanarity with the carboxylate--COO--atoms. The conformation of this molecule is compared with that of an analogous compound O-[3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oyl]-L- beta- phenyllactate, which has been employed as a spectroscopic substrate probe of carboxypeptidase A (L. C. Kuo, J. M. Fukuyama, and M. W. Makinen (1983) Journal of Molecular Biology 163, 63-105). The rotamer conformation of the free spin-label ester in solution, as determined in this study, and that of the enzyme-bound spin-labeled phenyllactate are compared. Differences in rotamer structure are discussed in terms of stereoelectronic principles that govern the pathway of substrate hydrolysis catalyzed by carboxypeptidase A.

Assignment of proton endor resonances of nitroxyl spin-labels in frozen solution.

Spin-label nitroxyl derivatives of tetramethylpyrroline and tetramethylpyrrolidine in frozen solutions of perdeuterated methanol have been characterized by electron nucleus double resonance (ENDOR spectroscopy). With use of selectively deuterated derivatives of 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide, proton ENDOR resonance features have been assigned to the vinylic proton in the five membered pyrrolinyl ring and to the methyl groups. The ENDOR resonance features were analyzed on the basis of their dependence on H0. Two pairs of resonance features were assigned to the vinylic proton and were shown to correspond to parallel and perpendicular hyperfine coupling (hfc) components. Six pairs of resonance features were ascribed to the methyl groups. The proton ENDOR spectra of the 3-carboxylic acid spin-label derivatives of tetramethylpyrroline and of tetramethylpyrrolidine compounds exhibited comparable features with nearly identical line splittings. From the observed ENDOR splittings, we have estimated the isotropic hfc component of the vinylic proton in 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide to be -1.81 +/- 0.04 MHz in frozen methanol. On the basis of the anisotropic dipolar hfc components, the electron-to-vinylic proton distance is estimated as 3.78 +/- 0.01 A, in excellent agreement with that of 3.79 A calculated from X-ray defined coordinates.