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L Prasad

Publications and source records attributed to L Prasad.

18 recordsLinked to original sources
PubMed1999

Conformational and quantitative structure-activity relationship study of cytotoxic 2-arylidenebenzocycloalkanones.

Various 2-arylideneindanones 1, 2-arylidenetetralones 2, and 2-arylidenebenzosuberones 3 were synthesized with the aim of determining the relative orientations of the two aryl rings which favored cytotoxicity. Molecular modeling of the unsubstituted compound in each series revealed differences in the spatial arrangements of the two aryl rings, and evaluation of these compounds against P388, L1210, Molt 4/C8, and CEM cells as well as a panel of human tumor cell lines indicated that in general the order of cytotoxicity was 3 > 2 > 1. In particular 2-(4-methoxyphenylmethylene)-1-benzosuberone (3k) had the greatest cytotoxicity, possessing 11 times the potency of the reference drug melphalan when all five screens were considered. Series 3 was considered in further detail. First, excision of the aryl ring fused to the cycloheptanone moiety in series 3 led to some 2-arylidene-1-cycloheptanones 4 which had approximately one-third of the bioactivity of the analogues 3. Second, in some screens cytotoxicity was correlated negatively with the sigma values and positively with the MR constants of the substituents in the arylidene aryl ring of 3. Third, X-ray crystallography of five representative compounds (3i,k-n) revealed differences in the locations of the aryl rings which may have contributed to the variations in cytotoxicity. Finally three members of series 3 inhibited RNA and protein syntheses and induced apoptosis in human Jurkat T cells. This study has revealed that 2-arylidene-1-benzosuberones are a group of useful cytotoxic agents, and in particular 3k serves as a prototypic molecule for subsequent structural modifications.

Animals↗
PubMed1999

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Amino Acid Sequence↗
PubMed1998

The 2.5 A resolution structure of the jel42 Fab fragment/HPr complex.

The tertiary structure of Jel42 Fab fragment complexed with HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli, has been determined at 2.5 A resolution. X-ray diffraction from a larger crystal provided 22,067 unique reflections as compared to 14,763 unique reflections (2.8 A resolution), which were obtained previously from a smaller crystal. The higher resolution allowed for more precise location of amino acid side-chains and for the location of well-ordered water molecules. Five more residues in the Fab fragment are found to be involved in binding HPr and two additional residues are identified as part of the epitope, bringing the totals to 24 and 16, respectively. At least nine water molecules are found at the interface between the two proteins, and these mediate hydrogen bonding interactions between the Fab fragment and HPr. Three additional hydrogen bonds have been identified (bringing the total to ten) and one salt-bridge occurs between LysL50 of the L2 complementarity-determining region (CDR) and GluP66 of HPr. This salt-bridge is the only interaction between HPr and CDRL2; thus all six CDRs are involved in binding. Inspection and empirical energy minimization of mutant HPrs in the complex indicate that, in some cases in the binding interaction, water molecules may compensate for residue alterations. Binding to the mutant SerP64Tyr HPr may require a movement of the HPr main chain. The active centre region of HPr, which is not involved in binding the antibody, and which was not resolved in the 2.8 A resolution structure of the complex, was determined. This active centre determined at pH 5.8, which is completely free of intermolecular contacts due to crystal packing, shows a potential hydrogen bond between the AsnP12 OD1 atom and the HisP15 NE2 atom, and no involvement of the C terminus with HisP15. The HisP15 ND1 atom is the site of phosphorylation in HPr. Although a specific amino acid at residue 12 is not conserved in HPr molecules from all species, a hydrogen bond between the side-chains of residue 12 and HisP15 may be a conserved feature of the active centres.

Antigen-Antibody Complex↗
PubMed1998

Conversion of an antibody into an enzyme which cleaves the protein HPr.

Jel 42 is an IgG which binds to the small bacterial protein, HPr and the structure of the complex is known at high resolution. The IgG was expressed as a single chain variable fragment (scFv) and the binding to HPr was assessed by fluorescence polarization of fluorescein-labelled HPr. The binding constant for the IgG was about 20-fold higher than the scFv. Inspection of the structure of the complex suggested that it might be possible to convert the scFv into a bond-specific protease by the introduction of three catalytic residues: a glutamate to increase the nucleophilicity of a nearby water molecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group. By trial and error it was found that a fourth residue had to be converted into glycine in order to maintain the integrity of complimentarity-determining region three of the heavy chain (CDRH3) at the binding interface. The resulting quadruple mutant still bound to HPr and unlike other mutants, showed weak protease activity as judged from the fluorescence polarization assay. The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue. With the aid of HPr fluorescein-labelled at two different positions, it was demonstrated that the size of the products was consistent with cleavage occurring in the vicinity of the target peptide bond. The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction.

Animals↗
PubMed1996

Complementarity determining region residues aspartic acid at H55, serine at H95 and tyrosines at H97 and L96 play important roles in the B72.3 antibody-TAG72 antigen interaction.

Structural analysis derived from the crystallographic study of the chimeric B72.3 antibody illustrated some major atomic interactions between complementarity determining region (CDR) residues. For example, hydrogen bonds are formed between H35/H95, L50/H97, H53/H55 and H96/L96 respectively. These CDR residues may play important roles in the B72.3-TAG72 (antibody-antigen) interaction either by direct interaction with the TAG72 antigen or by maintaining a CDR loop conformation through atomic interactions between CDR residues. In order to confirm these assumptions, we altered these CDR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen in a solid-phase radioimmunoassay. We found that H55, H95, H97 and L96 are important CDR residues for the B72.3-TAG72 interaction. Single amino acid substitutions of aspartic acid and serine by alanine at H55 of CDR2 and at H95 of CDR3 respectively and of tyrosine by phenylalanine at H97 and L96 of CDR3, significantly reduced the binding affinity for the TAG72 antigen by 20-, 8-, 16- and 45-fold respectively. Therefore, this study reveals some of the requirements for maintaining the integrity of the B72.3 antibody combining sites.

Amino Acid Sequence↗
PubMed1995

Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops.

Structural analysis derived from the crystallographic study of the chimeric B72.3 antibody illustrated that some heavy-chain framework residues having atomic interactions with heavy-chain CDR residues may directly affect the conformation of CDR loops. For example, an alanine residue at H71 provides room for packing CDR2/CDR1 and lysine residues at H73 and H93 contribute a salt-bridge to aspartic acid at H55 in CDR2 and a hydrogen bond to the carbonyl group at H96 in CDR3, respectively. We have analysed the contribution of these framework residues to the TAG72-binding affinity. We altered these framework residues by site-directed mutagenesis, and determined the affinity of these mutant chimeric antibodies for the TAG72 antigen by solid phase radioimmunoassay. We found that a single amino acid substitution of alanine by phenylalanine at H71 or lysine by isoleucine at H93, significantly reduced the binding affinity for the TAG72 antigen by 12 and 20-fold, respectively, whereas the substitution of lysine by alanine at H73 reduced the binding affinity only two-fold. Our results indicate that heavy-chain framework residues alanine at H71 and lysine at H93 of the chimeric B72.3 antibody are the major determinants of the conformation of heavy-chain CDR2/CDR1 and CDR3 loops, whereas the salt-bridge between lysine at H73 and aspartic acid at H55 is less important. The hydrogen bond between two framework residues, glutamine at H5 and serine at H25 does not affect any CDR conformation. Our results will thus be of importance especially when the humanized B72.3 antibody is constructed by grafting the CDR loops to a human framework. The important framework region interactions must be maintained in the final humanized antibody.

Amino Acid Sequence↗
PubMed1993

Evaluation of mutagenesis for epitope mapping. Structure of an antibody-protein antigen complex.

The location and description of epitopes on proteins describe the basis of immunological specificity. The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined. This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R. E., Delbaere, L. T. J., Lee, J. S., and Waygood, E. B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4877-4881), with those from x-ray analysis. There are 14 amino acid residues of E. coli HPr that interact with the Jel42 antigen-binding site. Nine of these were correctly assigned by the mutagenesis studies. Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent. Four residues were incorrectly assigned to the epitope. These residues were located adjacent to epitope residues that were likely perturbed by these mutations. This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incorrect assignments.

Amino Acid Sequence↗
PubMed1993

Structures of the lectin IV of Griffonia simplicifolia and its complex with the Lewis b human blood group determinant at 2.0 A resolution.

The structures of the fourth lectin isolated from Griffonia simplicifolia (GS4) and its complex with the methyl-glycoside of the Lewis b human blood group determinant (Le(b)-OMe) are reported at high resolution. The native GS4 crystal is isomorphous with the complexed GS4 crystal. The space group is P4(2)2(1)2 with unit cell dimensions a = 78.9 A, c = 89.1 A with one subunit of the lectin (bound to 1 Le(b)-OMe in the complex) in the crystallographic asymmetric unit. The native GS4 structure was solved by the molecular replacement technique and least-squares refined (PROLSQ and X-PLOR). The orientation of the Le(b)-OMe tetrasaccharide in the complex was established from a 2.8 A difference map with coefficients (Fcomplex--Fnative) and calculated phase angles from the native model. Both the final native and complex GS4 models consist of 1904 protein non-hydrogen atoms, one sulfate ion, one Ca ion, one Mn ion and three covalently-bound sugar residues N-linked to Asn18. In addition, the complex model has 47 Le(b)-OMe non-hydrogen atoms. The two structures have 135 water molecules in common in addition to eight and nine unique water molecules in the native and complex structures, respectively. The root-mean-square deviations from ideal bond distances and angles are 0.016 A, 3.2 degrees and 0.016 A, 3.0 degrees, for the native and complexed GS4, respectively. The R index for all unique data from 8 to 2.0 A is 0.187 for the native (19,204 reflections) and 0.181 for the complex (19,212 reflections). The tertiary structure of each subunit is similar to that of other leguminous lectins but the quaternary structure of the molecular dimer is different from that of any other lectin reported to date. The co-ordination about the Ca ion is pentagonal bipyramidal (with 1 long Ca(2+)-oxygen bond) and the co-ordination about the Mn ion is octahedral. Two conserved residues (Asp149 and Ser155) appear to be important because they are hydrogen-bonded to each other and to groups that co-ordinate the Mn ion. There are three cis-peptides in the polypeptide chain; two involve non-proline residues, one of which is homologous with other leguminous lectins and the other is unique to GS4. The two non-proline cis-peptides are located in the carbohydrate-binding site and are important for the specificity of the lectin. The molecular recognition of Le(b)-OMe by GS4 involves both polar and extensive non-polar interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1993

Inosithin neutralization test to measure lupus anticoagulants.

The inosithin neutralization test was performed in 14 patients in whom lupus anticoagulant was detected. To test its specificity, it was also performed in 10 patients with severe hemophilia and in three patients with Factor VIII inhibitors. Prolonged kaolin clotting time was corrected by adding varying amounts of inosithin (Asolectin, 0.19 to 100 micrograms), a soybean-derived phospholipid, in all patients with lupus anticoagulant but not in patients with hemophilia or in two patients with Factor VIII inhibitors. In one patient, both Factor VIII inhibitors and lupus anticoagulant were present. The concentration of lupus anticoagulant in a patient was expressed as the amount of inosithin (measured in micrograms) required to normalize the prolonged kaolin clotting time. This amount correlated significantly with the occurrence of thrombosis and/or recurrent abortion. The inosithin neutralization test is useful to measure lupus anticoagulant.

Adolescent↗
PubMed1991

Evaluation of four coagulation tests to detect plasma lupus anticoagulants.

Lupus anticoagulant was detected in 205 newly diagnosed, untreated patients with systemic lupus erythematosus by the following tests: kaolin clotting time, activated partial thromboplastin time, plasma prothrombin time, and, in the last 99 patients, by dilute Russell's viper venom time. In 10 patients, lupus anticoagulant was detected by kaolin clotting time prolongation, corrected by inosithin but not by normal plasma; 12 and 6 of them had prolonged activated partial thromboplastin time and partial plasma prothrombin time, respectively. Only 10 patients had a history of recurrent abortions and/or thrombosis, nine of whom had lupus anticoagulant as shown by the kaolin clotting time test. Of the 99 patients studied by all four tests, 9 showed lupus anticoagulant by both kaolin clotting time and dilute Russell's viper venom time; 7 had a history of abortion and/or thrombosis. The dilute Russell's viper venom time test is easy to perform and not affected by inhibitors to factor VIII or IX. It is recommended as a primary screening test for lupus anticoagulant detection in a hospital clinical laboratory.

Adolescent↗
PubMed1988

Structure determination of a monoclonal Fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.

Jel 42 is a monoclonal antibody specific for histidine-containing protein, a small phosphocarrier protein required for sugar transport in Escherichia coli. Fab fragments prepared from this antibody by papain digestion consisted of three major isoelectric forms which were separated on a chromatofocusing column. Two of these forms produced large crystals in space group P21 and unit cell dimensions a = 117.48 A, b = 66.56 A, c = 67.31 A, and beta = 118.7 degrees, with two Fab fragments per asymmetric unit. Data were collected to 3.5-A resolution. The structure of Fab Jel 42 was solved by the Molecular Replacement method (least-squares refined to R = 0.282) using the known structure of Fab HED 10 (12) as the search model; the amino acid residues of the hypervariable and elbow regions of Fab HED 10 were omitted from the starting model. A Fourier map calculated at this stage revealed electron density which corresponded to the hypervariable loops forming the antigen-binding crevice and the elbow region of Fab Jel 42. The elbow angles for the two independent Fab molecules are 159 and 167 degrees, similar to that of the Fab HED 10 search model which has an elbow angle of 162 degrees. There is no local noncrystallographic axis of symmetry relating the two molecules in the asymmetric unit.

Antibodies, Monoclonal↗
PubMed1986

Aging studies on fingerprint residues using thin-layer and high performance liquid chromatography.

The chemical substances of perspiration found in latent fingerprint residue may hold the key for dating latent fingerprints. To study the chemical transformations of these components as a function of time and environment, fingerprint residues from four males and four females were analysed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Three major and two or three minor components were detected by both methods and the variation in the quantity of the major components with time was observed.

Chromatography, High Pressure Liquid↗