Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm

The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.     

Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4-8 region

Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr(5), and the importance of positions 4 and 8. For example, when Trp(8) and Phe(4) were exchanged, the resulting analogue (Trp(4),Phe(8)-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe(4), with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp(8) (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp(8) (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.     

Studies in humans on the mechanism of potent spermicidal and apoptosis-inducing activities of vanadocene complexes

We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three complexes included vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC). The concentration-dependent effect of these vanadocenes on sperm-immobilizing activity (SIA), mitochondrial membrane potential (DeltaPsim), axonemal dynein ATPase activity, and tyrosine phosphorylation of global and axoneme-specific sperm proteins was assessed by computer-assisted sperm analysis, flow cytometry, colorimetry, and immunoblotting, respectively. Apoptosis-inducing ability was quantitated by the two-color flow cytometric terminal dideoxynucleotidyl transferase-based assay that labels 3'-hydroxyl ends of fragmented DNA. All three vanadocenes induced rapid sperm immobilization (T(1/2) < 15 sec). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC by 10-fold. The EC(50) values (50% inhibitory concentration) for VDC, VMDC, and VPMDC were 7.5 microM, 4.3 microM, and 0.7 microM, respectively. Whereas SIA of vanadocenes was apparent at low micromolar concentrations, the apoptosis-inducing property was evident only at higher micromolar concentrations. The concentrations of VDC, VMDC, and VPMDC required for 50% apoptosis were 49 microM, 67 microM, and 153 microM, and for 50% reduction in sperm DeltaPsim were 435 microM, 173 microM, and 124 microM, respectively. Spermicidal activity of vanadocenes was not dependent on the inhibition of ATPase or tyrosine phosphorylation of global and sperm axonemal proteins. Due to the ability of these vanadocene complexes to rapidly generate hydroxyl radicals in the presence of oxidant, our findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes gives them particular utility as a new class of vaginal contraceptives.     

Actions between neonicotinoids and key residues of insect nAChR based on an ab initio quantum chemistry study: hydrogen bonding and cooperative pi-pi interaction

Neonicotinoid insecticides show selective actions on insect nicotinic acetylcholine receptor (nAChR). Two key residues (Trp and Arg/Lys) have been identified as contributing to the neonicotinois binding. To investigate the selective mechanism, a computational model was set up to simulate the interaction between residues (Trp and Arg) of insect nAChR and neonicotinoids by quantum chemistry method. Three analogues of neonicotinoid derivatives without the chloropyridinyl moiety and 3-methyl-indole (3MI), guanidinium (Gua) were used to mimic the neonicotinoids and the side chain of key residues Trp and Arg accordingly. Interaction features of 3MI-analogues, analogues-Gua and 3MI-analogues -Gua complexes were analyzed comparatively. Hydrogen bonding between the nitro group of analogues and Gua was found to be the most important for binding. Moreover, the cooperative pi-pi interaction between analogues and the indole ring, which is strengthened by the existence of Gua, also contributes to the binding. The alternative binding model of neonicotinoids proposed here, although slightly different from others, might be close to the actual.     

The spermicidal and antitrichomonas activities of SSRI antidepressants

The study investigated spermicidal and antitrichomonas activities of selective serotonin reuptake inhibitor (SSRI) antidepressants with a view to generate new lead for development of dual-function spermicidal microbicides, which is an urgent global need. Fluoxetine, Sertraline, and Fluvoxamine exhibited both spermicidal and anti-STI (antitrichomonas) activities in vitro, whereas Paroxetine and Citalopram showed only the spermicidal activity. Fluoxetine exhibited better activity profile than the other antidepressant drugs with its spermicidal and antitrichomonas activities being comparable to that of the OTC contraceptive Nonoxynol-9. The non-detergent nature of Fluoxetine and a much lower spermicidal ED50 value (than N-9) may add considerably to its merit as a candidate for microbicidal contraceptive. Thus, the antidepressants exhibiting both spermicidal and antitrichomonas activities might provide useful lead for the development of novel, dual-function spermicidal contraceptives.     

Structure-activity studies on prolactin-releasing peptide (PrRP). Analogues of PrRP-(19-31)-peptide.

An investigation of a series of single replacement analogues of PrRP-(19-31)-peptide has shown that good functional activity was retained when Phe31 was replaced with His(Bzl), Phe(4Cl), Nle, Trp, Cys(Bzl) or Glu(OBzl); when Val28 or Ile25 was replaced with Phg; when Gly24 was replaced with D-Ala, L-Ala, Pro or Sar; when Ser22 was replaced with Gly and when Ala21 was replaced with Thr or MeAla. The results confirm that the functionally important residues are located within the carboxyl terminal segment, -Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2.     

Structure-activity relations in the oxidation of phenethylamine analogues by recombinant human liver monoamine oxidase A

The interaction of recombinant human liver monoamine oxidase A (MAO A) with a series of phenethylamine substrate analogues has been investigated by steady-state and stopped-flow kinetic techniques. Substrate analogues with para substituents exhibit large deuterium kinetic isotope effect on k(cat), on k(cat)/K(m), and on the limiting rate of enzyme reduction in reductive half-reaction experiments. These kinetic isotope effect values range from 5 to 10 with the exception of tyramine, which exhibited smaller steady-state isotope effects (2.3-3.5) than that observed on the rate of flavin reduction (6.9). The stopped-flow data show that imine release from the reduced enzyme is slower than the rate of catalytic turnover. Phenethylamine oxidation by MAO A can be described as the C-H bond cleavage step being rate limiting in catalysis and with oxygen reacting with the reduced enzyme-imine complex. In the case of tyramine, the product release from the oxidized enzyme-imine complex contributes to the rate limitation in catalysis. The binding affinities of a series of para-substituted phenethylamine analogues to MAO A show an increase in affinity of the deprotonated amine with increasing van der Waals volume of the substituent. The limiting rate of enzyme reduction decreases with increasing van der Waals volume of the substituent in a linear manner with no observable electronic contribution as observed previously with benzylamine reduction of MAO A [Miller, J. R., and Edmondson, D. E. (1999) Biochemistry 38, 13670-13683]. Examination of side chain analogues of phenethylamine show 3-phenylpropylamine to be oxidized 2.5-fold more slowly and bound 75-fold more tightly than phenethylamine. 4-Phenylbutylamine is not a substrate for MAO A but is a good competitive inhibitor with a K(i) value of 31 +/- 5 microM. Analysis of the effect of alkyl side chain alterations on binding affinities of a series of arylalkylamine analogues taken from this study and from the literature show a linear correlation with the Taft steric value (E(s)) of the side chain. These results suggest that the binding site for the aryl ring is identical for phenethylamine and for benzylamine analogues and that steric interactions of the alkyl side chain with the enzyme strongly contribute to the binding affinities of a series of reversible inhibitors of MAO A.     

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.     

Influence of long-term stability conditions on microbicidal nucleoside prodrug (WHI-07)-loaded gel-microemulsion

The objective of this study was to evaluate the long-term stability of the antiretroviral spermicide WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxyalaninyl phosphate) in a polymer-based microemulsion. The recovery and stability of WHI-07 in gelmicroemulsion was examined by a validated high-performance liquid chromatography (HPLC) method. The stability was examined over a period of 24 weeks at 3 controlled temperatures (4°C, 25°C, and 40°C). The recovery of the prodrug from 0.5% to 2.0% WHI-07-loaded gel-microemulsion was 99.8%. HPLC analysis revealed that a 2% WHI-07-loaded gel-microemulsion stored at room temperature and cold temperatures for 24 weeks retained >90% of the prodrug, whereas those stored at 40°C maintained 90% of initial WHI-07 for at least 10 weeks. The observed stability of WHI-07 in gel-microemulsion is of great importance for its widespread utility in various climatological conditions. Published: September 1, 2006     

Solution structure of Compstatin,a potent complement inhibitor.

The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13-residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure-function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry-restrained simulated-annealing methodology, using distance, dihedral angle, and 3J(NH-Halpha)-coupling constant restraints. The Compstatin structure contained a type I beta-turn comprising the segment Gln5-Asp6-Trp7-Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3J(NH-Halpha)-coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100-fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side-chain interactions and the beta-turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.     

Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability

Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents. Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase. Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells. These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity. All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability. These results and other structure-activity relationships [Baffi, R. A., Shenbagamurthi, P., Terrance, K., Becker, J. M., Naider, F., & Lipke, P. (1984) J. Bacteriol. 158, 1152-1156] indicate that the agglutination and morphological responses to alpha-factor can be varied independently. Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity. Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation. All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

Restricted rotation in chiral peptide nucleic acid (PNA) monomers--influence of substituents studied by means of 1H NMR

The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into "aminoalkyl" part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of 1H NMR at 0, 25 and 40 degrees C in CDCl3. The delta G0 values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.     

The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin.

We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin. We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins. All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9. Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa. The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM). The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity. All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).     

Conformation of the eosinophil chemotactic tetrapeptides and analogues in dimethyl sulfoxide. 1H n.m.r. studies

Proton nuclear magnetic resonance parameters are reported for DMSO-d6 solutions of the eosinophil chemotactic tetrapeptides, Val1-Gly2-Ser3-Glu4 and Ala1-Gly2-Ser3-Glu4, as well as three analogues of the Val1 tetrapeptide, D-Val1, Ala2 and Ala3. The synthesis of Val-(S)-[alpha-2 H1] Gly-Ala-Glu, in which the glycine has been stereospecifically deuterated in the H alpha 3 position, has allowed the assignment of the 1H resonances belonging to individual H alpha 2 and H alpha 3 glycine methylene protons. Simulation of the glycine ABX spin system yields two vicinal coupling constants which are consistent with a highly preferred conformation about the glycine HN-C alpha bond. The chemical shifts, coupling constants, temperature coefficients of amide proton chemical shifts and calculated side chain rotamer populations are reported for all peptides. The coupling constant analysis and temperature coefficients of amide proton chemical shifts together suggest that a type I beta-turn conformation is preferred by the Ala3 analogue. The 1H n.m.r. parameters of the other peptides suggest that these can also adopt a beta-turn conformation in DMSO. There are, however, considerable differences in the extent of conformational averaging undergone by the various peptides.