Anthony Tsarbopoulos

Ras oncoprotein inhibitors: The discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug-protein complex

The nucleotide exchange process is one of the key activation steps regulating the ras protein. This report describes the development of potent, non-nucleotide, small organic inhibitors of the ras nucleotide exchange process. These inhibitors bind to the ras protein in a previously unidentified binding pocket, without displacing bound nucleotide. This report also describes the development and use of mass spectrometry, NMR spectroscopy and molecular modeling techniques to elucidate the structure of a drug-protein complex, and aid in designing new ras inhibitor targets.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.

Isolation and characterization of a monomethioninesulfoxide variant of interferon α-2b

AbstractPurpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon α-2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN α-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN α-2b molecule. The far-UV CD spectra showed a slight loss of α-helical content and an increase in the β-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon α-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN α-2b variant was found to be present in the rHuIFN α-2b bulk drug substance in solution. The Met111 residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met111 residue did not seem to have a detectable effect on the biological activity of the molecule.

Glycoprotein analysis by delayed extraction and post-source decay MALDI-TOF-MS

Abstract Matrix-assisted laser desorption-ionization (MALDI)-MS analysis of glycoprotein samples is usually affected by metastable fragmentation, particularly in reflectron instruments. Use of delayed extraction (DE) is shown to minimize the observed metastable fragmentation. In the case of the CHO IL-4, which contains complex-type N-linked glycans, the disialylated component gives the most abundant MALDI signal with a dramatically improved resolution and mass accuracy over the non-DE linear TOF analysis. This increased resolution is advantageous for the 30 kDa Sf9-derived IL-4 receptor, where it is possible to differentiate the individual glycans, but not for higher mass and more heterogeneous glycoproteins. It is also shown that MALDI analysis of proteins with labile functional groups is more superior and reliable with linear DE-TOF systems rather than reflectron TOF analyzers. Further structural information on the sequence and disulfide mapping, as well as glycopeptide identification can be obtained by post-source decay (PSD) analysis of peptide and glycopeptide isolated fractions. The PSD formation of a characteristic triplet of ions separated by 33 Da in mass can be used to identify disulfide-paired peptides, even from complex digest mixtures of proteins.

Studies of the Ras-GDP and Ras-GTP noncovalent complexes by electrospray mass spectrometry

Abstract A novel MS based methodology utilizing electrospray ionization is described for the detection of the noncovalent interaction between a host protein (ras) and its guest ligands (GDP and GTP). The ras proteins are regulatory guanine nucleotide binding proteins which serve as signal transducers controlling cell proliferation or differentiation. They can exist in two interconvertible states of GDP-bound (inactive form) and GTP-bound (active form). The presence of the noncovalent complexes of ras-GDP and ras-GTP, as well as the unbound apo-ras protein, in various sample solutions containing biological buffers were confirmed by the observed average molecular weights of 19295, 19374, and 18852 Da, respectively. The stability of the observed GDP-bound and GTP-bound complexes is a combined function of solution pH and organic modifier content.

Chloramine T-induced structural and biochemical changes in echistatin.

Echistatin is a member of the disintegrin family of peptides and a potent inhibitor of platelet aggregation and cell adhesion. Echistatin binds to integrin αvβ3 and αIIbβ3 receptors with high affinity. Binding is mediated by an RGD‐containing loop maintained in an appropriate conformation by disulfide bridges. In this study, we have compared the binding characteristics of echistatin iodinated by either lactoperoxidase or chloramine T method. We show that echistatin labeled by lactoperoxidase method binds to integrin αvβ3 receptor with high affinity and in a non‐dissociable manner very similar to native echistatin. In contrast, chloramine T‐labeled echistatin can rapidly dissociate from the receptor. We demonstrate that chloramine T reaction results in the addition of an extra oxygen to the methionine residue adjacent to the RGD motif in echistatin. Modeling studies and molecular dynamic simulation studies show that the extra oxygen atom on the methionine residue can form hydrogen bonds with the glycine and aspartic acid residues of the RGD motif. These structural changes in echistatin help explain the changes in the binding characteristics of the molecule following chloramine T reaction.

Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry.

The structural characterization of the Escherichia coli-expressed human interferon alpha-2b (rh-IFN alpha-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods. The mass spectral data of the rh-IFN alpha-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule. The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN alpha-2b and the potent vasoconstrictor peptide endothelin.

Comparative studies of natural and recombinant proteins by californium-252 plasma desorption and cesium ion liquid secondary ion mass spectrometry

Abstract The relative molecular masses of five proteins in the 10–25 kDa range were successfully determined by 252 Cf plasma desorption mass spectrometry (PDMS) and 35-keV Cs + liquid secondary ion mass spectrometry (LSIMS). Both techniques were comparable in demonstrating high mass accuracy and sensitivity, although LSIMS required a shorter analysis time than PDMS. The sensitivity of these methods was excellent, requiring only low-nanomole amounts of protein. The determination of relative molecular mass by the plasma desorption nitrocellulose method was less affected by the salt and buffer content of the sample, as these components could be effectively removed by washing.

Demonstration of a 1–3 disulfide bond in a synthetic nonapeptide derived from the signal sequence and N-terminus of human γ-interferon

The nonapeptide Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys was prepared by solid-phase peptide synthesis under oxidizing conditions. Fast atom bombardment mass spectrometric analysis of the untreated molecule produced an ion consistent with a structure involving an intramolecular disulfide bond between Cys(1) and Cys(3). Mass spectra of the peptide after treatment with 2-mercaptoethanol gave signals corresponding to the reduced disulfide form of the peptide and to a mixed disulfide of the peptide with 2-mercaptoethanol. Molecular mechanics calculations of the conformation of the 11-membered ring formed by disulfide bond closure predicted a discrete, low-energy structure resembling the locus of a gamma turn. We hypothesize that this structure may be important in the recognition and cleavage of the signal sequence of the parent molecule.

Inactivation of the human cytomegalovirus protease by diisopropylfluorophosphate

Publisher Summary Cytomegalovirus (CMV) protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Ser132 as the active site serine. The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132 (DIP-CMV protease) is likely to resemble that of the tetrahedral transition-state intermediate. As the structure of the DFP-treated serine proteases resembles that of the tetrahedral transition-state intermediate, and the inactivated enzyme would not be susceptible to autoproteolysis, production of DIP-CMV protease would be useful for structure based drug design. The concentrations of DFP sufficient to yield stoichiometric incorporation of inhibitor at the active site of CMV protease, also resulted in substantial incorporation of DIP at a second site or sites. This heterogeneous incorporation would preclude crystallographic studies. For this reason, this chapter has attempted to optimize the conditions for inactivation of CMV protease with DFP, to produce pure DIP-CMV protease with minimum second site incorporation. Initial studies indicated that there was no loss of protease activity in the control samples, even when incubated for 23 hours at 22°C. A 3.8 hour incubation was sufficient to completely inactivate 180 μM protease in the presence of 4.3 mM DFP, while a 2.4 fold excess of the inhibitor inactivated only 50% of the enzyme. This indicates that CMV protease is less reactive with DFP than trypsin or chymotrypsin, and requires an order of magnitude excess of the organophosphate to achieve complete inactivation.