HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy.

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

Thermodynamics of peptide inhibitor binding to HIV-1 gp41.

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics. We have employed a soluble derivative of the gp41 core ectodomain and small cyclic disulfide D-peptide inhibitors to define the stoichiometry, affinity, and thermodynamics of ligand binding to this pocket using isothermal titration calorimetry. These inhibitors bind with micromolar affinity to the pocket with the expected stoichiometry of three peptides per gp41 core trimer. There are no cooperative interactions among the three binding sites. Linear eight- or nine-residue D-peptides derived from the pocket-binding domain of the cyclic molecules also bind specifically. A negative heat capacity change is observed and is consistent with burial of hydrophobic surface upon binding. Contrary to expectations for a reaction dominated by the classical hydrophobic effect, peptide binding is enthalpically driven and is opposed by an unfavorable negative entropy change. The calorimetry data support models whereby dominant negative inhibitors bind to a transiently exposed surface on the prefusion intermediate state of gp41 and disrupt subsequent resolution to the fusion-active six-stranded hairpin conformation.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

PSA-specific and non-PSA-specific conversion of a PSA-targeted peptide conjugate of doxorubicin to its active metabolites.

Tumor-selective delivery of doxorubicin by a prostate-specific antigen (PSA)-targeted peptide conjugate prodrug of doxorubicin was demonstrated in a nude mouse xenograft model of human prostate cancer. The prodrug (referred to as doxorubicin conjugate) contains doxorubicin linked to a seven-amino acid peptide conjugate that was designed to increase delivery of doxorubicin to tumor sites through the hydrolytic properties of PSA, which prostate tumors express in high amounts. Following i.p. administration of the doxorubicin conjugate to mice, tumor exposure to doxorubicin was increased 2.5-fold as compared with that achieved after an equimolar dose of doxorubicin itself. However, in heart tissue, the site of clinical dose-limiting toxicity, doxorubicin concentrations observed after administration of doxorubicin conjugate were substantially lower than those in mice that received doxorubicin itself. While the prodrug provided selective delivery of doxorubicin to tumor tissue, there was substantial non-PSA-specific formation of doxorubicin in laboratory animals, a factor that would limit the extent of therapeutic gain of the prodrug. Following i.v. administration to mice, rats, dogs, and monkeys, about one-third of the dose was metabolized to doxorubicin. In tumor-bearing mice, the fraction of the dose metabolized to doxorubicin appeared even higher. This is likely the result of conjugate conversion to doxorubicin by both PSA-specific (in tumor) and non-PSA-specific proteolytic activities. In vitro studies provided further support for the PSA specificity of metabolism; LNCaP cells mediated rapid metabolism of the conjugate, while DuPRO-1 cells, which are deficient in PSA, were incapable of metabolism.

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo.

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.

The significance of monoisotopic and carbon-13 isobars for the identification of a 19-component dodecapeptide library by positive ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

Harnessing the ultra high resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and positive ion electrospray, we have demonstrated the significance and utility of cumulative mass defect high resolution mass separation stable isotope distribution, exact mass measurement and elemental formula as a means of simultaneously identifying 19 components of the dodecapeptide library Ac-ANKISYQS[X]STE-NH(2). With an instrument resolution of 275 000 (average), isobaric multiplets attributed to monoisotopic and carbon-13 components of peptides: Ac approximately SLS approximately NH(2); Ac approximately SNS approximately NH(2); Ac approximately SOS approximately NH(2); Ac approximately SDS approximately NH(2); within the mass window of 1380-1385 Da, and Ac approximately SQS approximately NH(2); Ac approximately SKS approximately NH(2); Ac approximately SES approximately NH(2); Ac approximately SMS approximately NH(2), within the mass window 1395-1400 Da, were mass resolved, accurately mass measured and identified from the computed molecular formulas. This experimental procedure enabled the separation of monoisotopic and carbon-13 isobars yielding enhanced selectivity and specificity and serves to illustrate the significance of monoisotopic and carbon-13 isobars in final product analysis. Chromatographic separation (HPLC) was of limited utility except for monitoring the overall extent of reaction and apparent product distribution. Positive ion electrospray-FTICR-MS and fast atom bombardment (FAB) MS were used to assess final product quality and apparent component distribution.

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin.

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.

Structural studies on phospholamban and implications for regulation of the Ca(2+)-ATPase.

The cardiac sarcoplasmic reticulum (SR) protein phospholamban (PLB) is an endogenous inhibitor of the SR Ca(2+)-ATPase. Phosphorylation of PLB relieves this inhibition and up-regulates calcium transport. PLB has proved remarkably difficult to study by conventional solution-state nuclear magnetic resonance (NMR) methods, due primarily to the extreme hydrophobic nature of the protein and its propensity to form pentamers. That the C-terminal domain of PLB is helical and membrane spanning is now well established; the structure of the cytoplasmic domain is relatively ill defined. In order to discern the effect of phosphorylation on the structure of the cytoplasmic domain, we have characterized a variety of model peptides in several structure-inducing and/or lipid-mimicking environments using circular dichroism and solution-state NMR. The resolution of peptide structures obtained in aqueous trifluoroethanol was markedly improved by the incorporation of 15N labels into the peptide backbone, allowing a variety of isotope edited, filtered, and resolved techniques to be applied. Molecular dynamics simulations on the full-length protein were combined with an analysis of published data to suggest a revised model for the structure of PLB.

Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively dictated by those of the double mutants.

Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+.

The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for alpha-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) beta-hydroxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygenases.

Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.

Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies.

Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.

Identification and characterization of variants of tick anticoagulant peptide with increased inhibitory potency toward human factor Xa.

Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa (fXa), a central enzyme in the blood clotting cascade. As such, TAP is a potential antithrombotic agent. Site-directed mutagenesis studies were undertaken to determine the feasibility of increasing the inhibitory potency of TAP toward fXa. The amino acid substitutions Tyr-1 to Trp (Y1W) and Asp-10 to Arg (D10R) increased inhibitory potency toward human fXa by 2.5- and 4-fold, respectively. The increased inhibitory potency reflected a decrease in the rate constant for dissociation of the final fXa-TAP inhibitory complex. The double mutant, Y1W/D10R, exhibited an inhibition constant of 10 pM, a 37-fold enhancement of inhibitory potency toward human fXa. The improvement in inhibitory potency was less pronounced (12-fold) with dog fXa wherein Kis of 220 and 18 pM were observed for wild-type TAP and the double mutant, respectively. Mutation of Tyr-1 to Glu (Y1E) generated a weaker inhibitor (Ki = 2 nM) that bound human fXa more slowly. However, no change in inhibitory potency toward human fXa was observed when Tyr-1 was replaced by Phe. Taken together, these observations are consistent with the view that a hydrophobic amino acid at the N-terminus of TAP may be a determinant of inhibitory potency. Decreases by 3-4 orders of magnitude in inhibitory potency were noted upon mutation of Asn-2 and Leu-4 of TAP, further implicating the N-terminal domain as an important determinant of inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity studies of the s-echistatin inhibition of bone resorption.

Synthetic Arg-Gly-Asp (RGD)-containing peptides were examined in bone resorption or attachment and detachment assays with isolated mammalian osteoclasts in an effort to elucidate the mechanistic and structural basis for the inhibition of bone resorption by s-echistatin. Bone resorption was the process most sensitive to inhibition by s-echistatin, with IC50 = 0.3 nM; inhibition of attachment to bone or detachment (lamellipodial retraction) was 30- to 70-fold less sensitive, with IC50 = 10 or 20 nM, respectively. Single amino acid substitutions within the 49-residue sequence of s-echistatin showed that although the efficacy of s-echistatin is dependent on the Arg24-Gly25-Asp26 sequence, additional residues, including Asp27, Met28, and Cys39, are also critical for potent inhibition of the resorbing activity of isolated rat osteoclasts. Because of the identification of the av beta 3 as the primary integrin on rat osteoclasts interacting the RGD peptides (Helfrich et al.), we examined the possibility of modeling bone resorption with other beta 3-mediated processes. Specifically, av beta 3 endothelial cell (human or rat) attachment to vitronectin and aIIb beta 3 platelet aggregation were compared with bone resorption for sensitivity to s-echistatin analogs, linear RGD peptides, and cyclic RGD peptides. Essentially no similarity in sensitivity to RGD peptides were observed between bone resorption, platelet aggregation, or endothelial cell attachment. Because rat osteoclasts and human giant cell tumors (osteoclastomas) shared similar sensitivity to s-echistatin and rat and human endothelial cells showed a similar sensitivity profile to RGD peptides, the dissimilarity of bone resorption to other beta 3-mediated processes cannot be explained in terms of species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional structure of echistatin and dynamics of the active site.

The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly5 to Cys20 and from Asp30 to Asn42 has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted beta-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by 15N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3-0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop 'wagging' motional model, suggested by an examination of superposed solution structures.

Development of 1,4-benzodiazepine cholecystokinin type B antagonists.

A series of 3-(arylureido)-5-phenyl-1,4-benzodiazepines, nonpeptidal antagonists of the peptide hormone cholecystokinin (CCK), are described. Derived by reasoned modification of the CCK-A selective 3-carboxamido-1,4-benzodiazepine, MK-329, this paper chronicles the development of potent, orally effective compounds in which selectivity for the CCK-B receptor subtype was achieved. The principal lead structure that emerged from these studied is L-365,260, a compound which has been submitted for clinical evaluation. Details of the ability to modulate the receptor interactions of these benzodiazepines by appropriate structure modifications are discussed which imply the possibility of further refining the CCK-B receptor affinity and selectivity of this class of compounds.

Inhibition of osteoclastic bone resorption in vivo by echistatin, an "arginyl-glycyl-aspartyl" (RGD)-containing protein.

Osteoclastic bone resorption requires the formation of a tightly sealed compartment between the osteoclast and the mineralized bone matrix. This compartment functions as an extracellular "lysosome" which contains proteolytic enzymes and acids. Vitronectin receptors (VnR, integrin alpha v beta 3) displayed on the osteoclast cell surface may play a role in the attachment of osteoclasts to the resorption surface. VnR are known to bind to arginyl-glycyl-aspartyl (RGD)-containing matrix proteins and it has recently been reported that soluble peptides containing RGD sequences can block osteoclast attachment to bone and inhibit bone resorption in vitro. In this study echistatin, a naturally-occurring protein containing an RGD-sequence motif, was shown to completely inhibit osteoclast-mediated bone resorption in vivo. Echistatin or smaller derivative peptides may prove useful in the treatment of disorders characterized by excess bone resorption, such as osteoporosis and metastatic bone disease.

Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.