Isolation, identification and synthesis of locustamyotropin (Lom-MT), a novel biologically active insect peptide

A peptide that stimulates the spontaneous contractions of the hindgut of Leucophaea maderae has been purified from extracts of brain-corpora cardiaca/corpora allata-subesophageal ganglion complexes of 9000 adult Locusta migratoria and was designated locustamyotropin or Lom-MT. The primary structure of this 12 residue peptide has been determined Gly-Ala-Val-Pro-Ala-Ala-Gln-Phe-Ser-Pro-Arg-Leu-NH2. The C-terminal sequence (Phe-Ser-Pro-Arg-Leu-NH2) is identical to the C-terminal pentapeptide of the pheromone biosynthesis activating neuropeptide, recently isolated from Heliothis zea, and is also similar to the C-terminal of leucopyrokinin of Leucophaea. Synthetic Lom-MT showed biological as well as chemical characteristics, indistinguishable from those of native Lom-MT. In locust preparations, Lom-MT provoked an increase in frequency, amplitude and tonus of contractions of the oviduct, but was inactive in the same conditions on the locust hindgut preparation.     

Microwave expedited synthesis of 5-aminocamptothecin analogs: Inhibitors of hypoxia inducible factor HIF-1alpha

A series of 5-aminosubstituted camptothecin analogs were prepared from the corresponding 5-hydroxycamptothecin using microwave irradiation. The analogs were assayed for ability to inhibit the action of hypoxia inducible factors (HIF-1alpha and HIF-2alpha). The 5-fluoroethyl analog showed potent inhibitory activity and is now the focus of ongoing pathway analysis and potential as an antiproliferative agent.     

The effect of proctolin analogues and other peptides on locust oviduct muscle contractions

The locust oviduct bioassay system was used to assess the ability of a variety of peptides to induce oviducal contractions. Proctolin analogues were three orders of magnitude less potent than proctolin. Proctolin supra-analogue and Arg-Tyr-Leu-Ala-Thr demonstrated high activity. Perhaps the most significant finding was the discrepancy between the high binding capacity of the proctolin analogue Arg-Tyr-Ser-Pro-Thr and its relatively low myotropic activity. This observation argues for a crucial role for the leucine residue in activating the proctolin receptor. Several other myotropic peptides were tested for their effect on oviduct contractions. FMRFamide caused contractions at doses several orders of magnitude higher than proctolin. The FLRFamide leucomyosuppressin inhibited proctolin-induced contractions. In addition, myomodulin and catch relaxing peptide caused oviducal contractions at low concentrations. The enkephalins had no effect when applied alone but potentiated proctolin-induced oviduct contractions. The mechanism of the potentiation is not known. The data argue for the presence of several binding sites on the oviduct membrane.     

Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins

Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata from the cockroach Diploptera punctata showed that the activity of analog I (IC₅₀: 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr-Xaa-Phe-Gly-Leu-NH₂, IC₅₀: 0.13 μM) it mimicked and the activity of the analog II (IC₅₀: 0.13 μM) proved roughly equivalent to the C-terminal pentapeptide. The results indicate that a new simple mimicry for Tyr-Xaa-Phe-Gly has been discovered; analog I may be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST analogs for insect management.     

Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala. Eight different analogs of the insect kinin C-terminal pentapeptide active core in which the critical residues Phe 1, Pro3 and Trp 4 are replaced with beta 3-amino acid and/or their beta2-amino acid counterparts were evaluated on recombinant insect kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini) and the dengue vector, the mosquito Aedes aegypti (L.). A number of these analogs previously demonstrated enhanced resistance to degradation by peptidases. Single-replacement analog beta 2 Trp 4 and double-replacement analog [beta 3 Phe 2, beta 3 Pro 3] of the insect kinins proved to be selective agonists for the tick receptor, whereas single-replacement analog beta 3 Pro 3 and double-replacement analog [beta 3 Phe, beta 3 Pro 3] were strong agonists on both mosquito and tick receptors. These biostable analogs represent new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin-regulated processes.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)

The systematic analysis of structure-activity relationships of insect kinins on two heterologous receptor-expressing systems is described. Previously, kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the dengue vector, the mosquito Aedes aegypti (L.), were functionally and stably expressed in CHO-K1 cells. In order to determine which kinin residues are critical for the peptide-receptor interaction, kinin core analogs were synthesized as an Ala-replacement series of the peptide FFSWGa and tested by a calcium bioluminescence plate assay. The amino acids Phe(1) and Trp(4) were essential for activity of the insect kinins in both receptors. It was confirmed that the pentapeptide kinin core is the minimum sequence required for activity and that the C-terminal amide is also essential. In contrast to the tick receptor, a large increase in efficacy is observed in the mosquito receptor when the C-terminal pentapeptide is N-terminally extended to a hexapeptide. The aminoisobutyric acid (Aib)-containing analog, FF[Aib]WGa, was as active as superagonist FFFSWGa on the mosquito receptor in contrast to the tick receptor where it was statistically more active than FFFSWGa by an order of magnitude. This restricted conformation Aib analog provides information on the conformation associated with the interaction of the insect kinins with these two receptors. Furthermore, the analog FF[Aib]WGa has been previously shown to resist degradation by the peptidases ACE and nephrilysin and represents an important lead in the development of biostable insect kinin analogs that ticks and mosquitoes cannot readily deactivate.     

Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.     

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.     

Design,synthesis and aphicidal activity of N-terminal modified insect kinin analogs

The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH₂. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH₂ (LC₅₀ = 0.045 mmol/L) was similar to the lead compound (LC₅₀ = 0.048 mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides.     

Molecular correlates of the action of bis(ethyl)polyamines in breast cancer cell growth inhibition and apoptosis.

Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50 values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11-bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 microM. Another group of three compounds showed antiproliferative activity at about 5 microM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.     

Roles of Individual Disulfide Bridges in the Conformation and Activity of μ-Conotoxin GIIIA,a Peptide Blocker of Muscle Sodium Channels

Seven analogs of μ-conotoxin GIIIA (μ-GIIIA), a specific blocker of muscle sodium channels, were synthesized by replacing stepwise the three cystine residues with Ala. The circular dichroism spectra of the analogs suggested that the deletion of disulfide bonds gradually randamized a conformation. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the deletion of one disulfide bond reduced the potency to less than 1 % of control. Monocyclic analogs and a linear analog were almost inactive. Therefore, all three disulfide bridges are essential for stabilizing the specific conformation of μ-GIIIA to show biological activity.     

Effect of three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 on 25-OH-D3-1 alpha-hydroxylase in isolated mitochondria and on 25-hydroxyvitamin D3 metabolism in cultured kidney cells

Three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)--2-nor-1,3-seco-1,25(OH)2D3 (2-nor analog), 2-oxa-3-deoxy-25-OH-D3 (2-oxa analog), and A-homo-3-deoxy-3,3-dimethyl-2,4-dioxa-25-OH-D3 (A-homo analog)--were tested for their ability to inhibit 25-OH-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) in isolated mitochondria and to alter 25-OH-D3 metabolism in cultured chick kidney cells. The 2-nor and 2-oxa analogs were relatively potent (Kis of 60 and 30 nM, respectively, compared with 170 nM for 1,25(OH)2D3), whereas the A-homo analog was completely ineffective in inhibiting 1 alpha-hydroxylase activity. In contrast, all three analogs were able to repress 1 alpha-hydroxylase and induce 24-hydroxylase activity in cultured chick kidney cells, suggesting that this process is not one of direct action in the mitochondria, but is more likely to be a receptor-mediated one.     

Influence of cyclooxygenase inhibitors and arachidonic acid on contractile activity of the human Fallopian tube

Small muscle strips were dissected from the circular and longitudinal muscle layers of the human oviduct. The preparations showed rhythmic spontaneous activity when perfused by Krebs-Ringer buffer. Excitatory effects of the prostaglandin (PG) precursor arachidonic acid were totally blocked by the cyclooxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and indomethacin. The latter drugs also caused a reversible inhibition of spontaneous activity in both muscle layers. After total inhibition produced by ETYA, the initial activity was restored by adding low concentrations of prostaglandin F2 alpha (PGF2 alpha) to the medium. PGE2 was able to reestablish the activity only in the longitudinal layer. It is concluded that isolated smooth muscle of the human oviduct has the capacity of generating PGs from both endogenous and exogenous substrate. The data also suggest that the formation of PGF2 alpha is a prerequisite for maintenance of normal tubal contractions.     

A review of the involvement of proctolin as a cotransmitter and local neurohormone in the oviduct of the locust,Locusta migratoria

The pentapeptide proctolin, originally identified in the cockroach, has been shown to be widely distributed in many insects and to have a broad range of physiological functions. In the oviduct of the locust, Locusta migratoria, proctolin's role as a neurotransmitter/neuromodulator has been well documented; however, a neurohormonal role in the locust is less certain. This review will examine the various roles of proctolin in locust oviduct contraction and will present evidence that a substance chromatographically, immunologically and physiologically indistinguishable from proctolin is present in the hemolymph of the locust, L. migratoria. This material is concentrated in the plasma, rather than the hemocytes, and is present at concentrations ranging from 0.1 to 0.2nM. This review extends the role of proctolin in insects, and suggests that proctolin may play a neurohormonal role in the locust.     

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.     

Relationship between structure and P-glycoprotein inhibitory activity of dimeric peptides related to the Dmt-Tic pharmacophore

To develop novel inhibitors of P-glycoprotein (P-gp), dimeric peptides related to an opioid peptide containing the Dmt-Tic pharmacophore were synthesized and their P-gp inhibitory activities were analyzed. Of the 30 analogs synthesized, N(α),N(ε)-[(CH(3))(2)Mle-Tic](2)Lys-NH(2) and its D-Lys analog were found to exhibit potent P-gp inhibitory activity, twice that of verapamil, in doxorubicin-resistant K562 cells. Structure-activity studies indicated that the correct hydrophobicity and spacer length between two aromatic rings are important structural elements in this series of analogs for inhibition of P-gp.     

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.     

Proctolin's role in neurally evoked contractions of the locust oviducts

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10⁻⁹ M and maxima at 2.5 × 10⁻⁸ M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 139–150, 1997     

Comparison of the myotropic activity of position-2 modified analogues of proctolin on the hindgut of Periplaneta americana and the oviduct of Locusta migratoria

The largest series of position-2 modified proctolin analogues to have been examined to date were tested for their ability to mimic the basal contraction induced by proctolin on hindgut of the cockroach, Periplaneta americana, and oviduct of the locust, Locusta migratoria. Twelve analogues of proctolin (Arg-Tyr-Leu-Pro-Thr), differing in the substituent (H, OMe, OEt, OPr, F, Cl, Br, I, NO(2), NH(2), N(3), Me) located at the para-position of the aromatic amino acid, caused dose-dependent contractions of both tissues at concentrations quite similar to proctolin. Seven showed greater or equal potency on the hindgut but, with one exception, they were less active on the oviduct than proctolin. The rank order of potency of the analogues depends on the tissue, lending more support to the notion that insects have more than one type of proctolin receptor. No relationship was observed between myoactivity and lipophilic, steric, electron donating or electron withdrawing properties of the substituents at the para-position of the aromatic amino acid. This may be the result of more than one sub-type of proctolin receptor on the specific tissue with differing structural requirements for optimum activity.