Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosol.

We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

Leptin-based glycopeptide induces weight loss and simultaneously restores fertility in animal models.

AIM: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. METHODS: The glycopeptide containing two additional non-proteinogenic amino acids was synthesized by standard solid-phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side-by-side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. RESULTS: The 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca) was designed to target a principal leptin/ObR-binding interface. E1/Aca induced leptin effects in ObR-positive cell lines at picomolar concentrations and readily crossed the blood-brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin-dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high-fat peanut diet in a dose-dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity-related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide-, but not leptin-treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. CONCLUSIONS: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin-deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.

A68: a major subunit of paired helical filaments and derivatized forms of normal Tau.

Putative Alzheimer disease (AD)-specific proteins (A68) were purified to homogeneity and shown to be major subunits of one form of paired helical filaments (PHFs). The amino acid sequence and immunological data indicate that the backbone of A68 is indistinguishable from that of the protein tau (tau), but A68 could be distinguished from normal human tau by the degree to which A68 was phosphorylated and by the specific residues in A68 that served as phosphate acceptors. The larger apparent relative molecular mass (Mr) of A68, compared to normal human tau, was attributed to abnormal phosphorylation of A68 because enzymatic dephosphorylation of A68 reduced its Mr to close to that of normal tau. Moreover, the LysSerProVal motif in normal human tau appeared to be an abnormal phosphorylation site in A68 because the Ser in this motif was a phosphate acceptor site in A68, but not in normal human tau. Thus, the major subunits of a class of PHFs are A68 proteins and the excessive or inappropriate phosphorylation of normal tau may change its apparent Mr, thus transforming tau into A68.

An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants.

Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.

Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects.

Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested.

A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2'-deoxyuridine 5'-triphosphates.

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase alpha and beta of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n-alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [3H]dAMP or [3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, alpha-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n-alkyl-dUTPs than beta-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2-14C]isopropyl-dUTP.

Glycosylation of synthetic T helper cell epitopic peptides influences their antigenic potency and conformation in a sugar location-specific manner.

The immunodominant T helper cell epitopes 31D and VF13N of rabies virus nucleoprotein and glycoprotein, respectively, correspond to peptide sequences AVYTRIMMNGGRLKR and VVEDEGCTNLSGF, and are expressed between amino acids 404-418 and 29-41, of the appropriate proteins. We investigated how internal or external glycosylation affects the biological activity and conformation of the peptides 31D and VF13N. Mid-chain incorporation of maltobiose or N-acetylglucosamine moieties into the asparagine residues greatly diminished the T-cell stimulatory activity in vitro (due to the diminished ability of the glycopeptides to bind to major histocompatibility complex determinants) and reduced the characteristic alpha-helicity of the peptides in aqueous trifluoroethanol solutions. In contrast, addition of maltobiose- or N-acetylglucosamine-coupled asparagines to the N-termini of peptides 31D and VF13N resulted in unchanged T-cell activity. Furthermore, N-terminal glycosylation of peptide 31D, as indicated by the functional assay, decreased the sensitivity of the peptide to degradation in human serum and did not affect the alpha-helical conformation. These data indicate that glycosylation of T-cell epitopes is not a preferable method for the preparation of antagonists, but incorporation of the sugars to appropriate positions may be advantageous in the design of T-cell agonists and peptide-based vaccines.

The effects of post-translational side-chain modifications on the stimulatory activity, serum stability and conformation of synthetic peptides carrying T helper cell epitopes.

Peptides 31D and VF13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68-76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55-64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, beta-linked sugars in mid-chain positions had a greater inhibitory effect than alpha-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the beta-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of beta-linked carbohydrates might be superior to the addition of alpha-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant alpha-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the alpha- and beta-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.

Comparison of the effects of amino acid substitutions and beta-N- vs. alpha-O-glycosylation on the T-cell stimulatory activity and conformation of an epitope on the rabies virus glycoprotein.

The first potential N-glycosylation site of the rabies virus glycoprotein, the antigen that carries epitopes for glycoprotein-specific T-cells and virus neutralizing antibodies, is glycosylated inefficiently. Recently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished the T-cell stimulatory activity and reduced the characteristic alpha-helicity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strains and appropriate tridecapeptide sequences and their analogs was investigated. The T-cell recognition pattern of the rabies and rabies-related viruses was identical to that of the synthetic peptides representing the respective epitope sequences. While the asparagine could be replaced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-galactosamine moiety at the serine, or galactosyl-N-acetyl-galactosamine moieties at the threonines preceding or replacing the asparagine (all O-linked sugars in the natural alpha-anomeric configuration) resulted in epitopes that lowered rather than abolished the T-cell stimulatory activity. All non-glycosylated peptides assumed a low-to-medium helicity in trifluoroethanol. O-glycosylation was more efficient than N-glycosylation in breaking the helical conformation of the peptides to result in the formation of reverse-turns or unordered structure.

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Metal ion-induced conformational changes of phosphorylated fragments of human neurofilament (NF-M) protein.

The NF-M subunit of human neurofilaments has a C-terminal repeating 13-mer sequence. The 13-mer (Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) (NF-M13) and 17-mer (Glu-Glu-Lys-Gly)-(NF-M13) sequences were synthesized, as were both the mono- and diphosphorylated Ser species. Circular dichroism (c.d.) studies and c.d. titrations with Al3+ and Ca2+ were performed. The conformation of the phosphorylated and unphosphorylated material was random in water. Deconvolution of the c.d. spectra, in trifluoroethanol, of the untitrated samples yielded a high content of unordered structure, similar to the poly-L-proline II structure. Titration of the phosphorylated species with Al3+ or Ca2+ caused a surprising conformational change to occur, yielding a high content of beta-pleated sheet structure. A mechanism of metal binding to the phosphofragments is proposed which may be relevant to the formation of neurofibrillary tangles in Alzheimer's disease.

Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant.

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by 1H-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 A and 0.66 A, respectively. The structures consist of two short antiparallel beta-strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta-strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides.

The adipokine leptin mediates muscle- and liver-derived IGF-1 in aged mice.

Muscle- and liver-derived IGF-1 play important roles in muscle anabolism throughout growth and aging. Yet, prolonged food restriction is thought to increase longevity in part by lowering levels of IGF-1, which in turn reduces the risk for developing various cancers. The dietary factors that modulate IGF-1 levels are, however, poorly understood. We tested the hypothesis that the adipokine leptin, which is elevated with food intake and suppressed during fasting, is a key mediator of IGF-1 levels with aging and food restriction. First, leptin levels in peripheral tissues were measured in young mice fed ad libitum, aged mice fed ad libitum, and aged calorie-restricted (CR) mice. A group of aged CR mice were also treated with recombinant leptin for 10 days. Later, aged mice fed ad libitum were treated with saline (VEH) or with a novel leptin receptor antagonist peptide (Allo-aca) and tissue-specific levels of IGF-1 were determined. On one hand, recombinant leptin induced a three-fold increase in liver-derived IGF-1 and a two-fold increase in muscle-derived IGF-1 in aged, CR mice. Leptin also significantly increased serum growth hormone levels in the aged, CR mice. On the other, the leptin receptor antagonist Allo-aca did not alter body weight or muscle mass in treated mice compared to VEH mice. Allo-aca did, however, produce a significant (20%) decline in liver-derived IGF-1 as well as an even more pronounced (>50%) decrease in muscle-derived IGF-1 compared to VEH-treated mice. The reduced IGF-1 levels in Allo-aca treated mice were not accompanied by any significant change in growth hormone levels compared to VEH mice. These findings suggest that leptin receptor antagonists may represent novel therapeutic agents for attenuating IGF-1 signaling associated with aging, and could potentially mimic some of the positive effects of calorie restriction on longevity.

Insect peptides with improved protease-resistance protect mice against bacterial infection.

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Quantification of modified amyloid beta peptides in Alzheimer disease and Down syndrome brains.

To gain insights into the different forms of modified amyloid beta peptides (A beta) in the Alzheimer disease (AD) and Down syndrome (DS) brain, we used two-site ELISAs with antibodies specific for isomerized (i.e. A beta with L-isoaspartate at positions 1 and 7) and pyroglutamate-modified (i.e. A beta beginning with pyroglutamate at position 3) forms of A beta to quantitate the levels of these different A beta peptides in formic acid extracts of AD and DS frontal cortex. Despite variations in the proportions of distinct forms of A beta in AD and DS frontal cortex, the major species of A beta in these samples were A betaN3(pyroGlu)-42 as well as A beta x-42 (where x is a residue at position 2 or less in A beta), whereas isomerized A beta was a minor species. Further, the levels of isomerized and pyroglutamate-modified forms of A beta terminating at amino acid 42 were higher than those ending at amino acid 40. The abundance of the distinct forms of A beta reported here in formic acid extracts of AD and DS frontal cortex suggests that these A beta species could play important roles in the deposition of A beta in AD and DS brains.

Spectroscopic evidence that monoclonal antibodies recognize the dominant conformation of medium-sized synthetic peptides.

Spectroscopic methods have amply documented that small- and medium-sized peptides tend to assume unordered conformations in water. The conformational tendencies, however, manifest in halogenated alcohols, and the preferred secondary structures are apparent from the circular dichroism (CD) spectra. Here we report the results of immobilizing peptide and protein antigens from various mixtures of trifluoroethanol and water during enzyme-linked immunosorbent assays. The increased recognition by the appropriate monoclonal antibodies (mAbs) is correlated with the increase of the alpha helical, beta turn, or beta pleated sheet content of the peptides presented in the different solvent mixtures. Remarkably, the antibody binding can be detected at considerably lower antigen levels if the antigen is immobilized from trifluoroethanol. The antigens we used corresponded to fragments of normal human neurofilaments and tau protein found in the paired helical filaments of Alzheimer's disease, and the nucleoprotein of rabies virus. The conformation of myoglobin is as stable in water as in trifluoroethanol, and therefore acted as a negative control. Indeed, the recognition of myoglobin did not increase upon increasing the trifluoroethanol concentration in the solvent used to apply the antigen to the plate. The possibility of imperfect binding to the plastic carrier or nonspecific binding to irrelevant antibodies is excluded by using control experiments. We offer the first direct evidence that the mAbs recognize the secondary structure of epitopes, and that it is possible to correlate the binding conformation of the epitopes with CD measurements made in trifluoroethanol-water mixtures.

In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen.

Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90x130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 microgram (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium-splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.

Intracellular targets of antibacterial peptides.

The recent past witnessed a decrease in the number of new antibacterial compounds approved by the regulatory agencies and an almost complete lack of molecules killing bacteria by novel mechanisms of action. The broad spectrum antimicrobial agents currently on the market carry the potential, and indeed victims, of resistance developed against them. The need for new types of antimicrobial drugs coincides with the desire of developing lead molecules that act selectively on a single strain, or perhaps on a few closely related strains. Such selectivity would exclude the likelihood of the emergence of broad-range resistance. Intracellular bacterial targets most prevalently proteins needed for the life cycle of bacteria, carry the potential to be a resourceful target for a new family of antimicrobial compounds. Inhibition of proteinaceous functions requires stereospecificity, and a drug structurally similar to the target proteins themselves. Indeed, some antibacterial peptides show selective inhibition of intracellular targets. A few native peptides and their designed analogs appear to kill only a limited number of bacterial strains. Identification of the binding sites on the target proteins would allow the design of strain-specific antibacterial and antifungal peptides without the fear of development of common resistance to these agents.

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.

Oxazepam esters. 2. Correlation of hydrophobicity with serum binding, brain penetration, and excretion.

Pharmacokinetics of a series of prodrug-type oxazepam esters were studied in mice. The effect of hydrophobicity was investigated in relation to serum binding, brain penetration, tissue storage, and excretion. Binding to mouse serum and to human serum albumin was measured by equilibrium dialysis, and the changes in binding free energy were correlated with RM values. Brain-blood partition of the esters did not change parallel with their serum binding. An indirect correlation exists between RM of the esters and oxazepam brain accrual. Brain-blood concentration ratios of oxazepam prove that hydrolysis precedes brain penetration and hydrophobicity might primarily influence the hydrolysis rate. The amount of tissue storage and total excretion rates also correlate with hydrophobicity.