John M. Stewart

The role of enzymatic processing in the biological actions of substance P

There is considerable evidence that substance P (SP) is a neurotransmitter in the CNS. Current findings suggest that the effects of synaptically released SP are terminated by enzymatic breakdown, primarily by endopeptidase 3.4.24.11 (endo 24.11). The products of cleavage by endo 24.11 include the amino-terminal fragment SP(1-7). Evidence suggests that SP is involved in mediating baroreceptor reflex activity in the nucleus of the solitary tract (NTS). Microinjection of SP into the NTS lowered blood pressure and heart rate. Microinjection of SP(1-7) into the NTS reproduced the effects of SP on both heart rate and blood pressure. Intra-NTS injection of phosphoramidon, an inhibitor of endo 24.11 activity, completely blocked the effects of a subsequent injection of SP. This blocking effect of phosphoramidon was unaltered by pretreatment with the opiate inhibitor naloxone. In contrast, phosphoramidon failed to block the depressor and bradycardic effects of SP(1-7). The implications of these findings regarding the role of endo 24.11 in the metabolism of SP are discussed.

Effects of substance P and neurokinin A (substance K) on motor behavior: unique effect of substance P attributable to its amino-terminal sequence.

The effects of intraventricular injections of the neuropeptides substance P (SP) and neurokinin A (NK-A; also called substance K) on spontaneous motor behavior were examined in mice. SP and NK-A were essentially equipotent at enhancing grooming and scratching behavior, and at reducing sniffing behavior. However, SP significantly enhanced hindlimb rearing behavior, while NK-A reduced this behavior. The effects of 3 other tachykinins, physalaemin, eledoisin and kassinin, were comparable to those of NK-A, including the reduction in rearing. Thus, SP is unique among tachykinins in its potentiation of rearing behavior. It was further demonstrated that carboxy-terminal SP fragments with tachykinin activity on smooth muscle resemble NK-A, and not SP, in their effects on motor behavior. In contrast, amino-terminal SP fragments, devoid of tachykinin-like activity, reproduced the one motor effect unique to SP, enhanced rearing, while lacking those actions common to all tachykinins. The structural requirements for enhanced rearing behavior by amino-terminal fragments were quite specific, in terms of chain length and sensitivity to D-amino acid substitutions, with the natural amino-terminal hexa- and heptapeptides being most active. The implications of these findings are discussed in light of recent observations that these same amino-terminal SP fragments are produced in vivo as metabolites of SP.

Treatment of severe pulmonary hypertension: a bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy.

Bradykinin is an important modulator of endothelial cell function and has also a powerful cardioprotective effect. Here we report that treatment of severely pulmonary hypertensive rats (that recapitulate several of the physiological and pathological characteristics of the human pulmonary vascular disease, including dramatic right ventricular hypertrophy, pericardial effusion and death) with a newly synthesized long-acting bradykinin B2 receptor agonist B9972 caused reduction of the pulmonary artery pressure (PAP=51+/-2.0 versus 68+/-2.8 of untreated animals) and of right ventricular hypertrophy (Rv/Lv+S=0.55+/-0.02 versus 0.73+/-0.03 of untreated rats) and activation of Akt. Long-term stimulation with B9972 in our animal model of SPH resulted in decreased expression of the B2 receptor in lung vasculature. Treatment with B9972 decreased the number of plexiform lesions in the lungs by inducing cell apoptosis in the obliterated vessels and by restoring caveolin-1 expression. B9972 also promoted eNOS activation. In vitro B9972 caused activation of caspase-3 as well as Erk and induction of prostacyclin production in rat pulmonary microvascular EC. Taken together our data suggest that a stable bradykinin B2 agonist B9972 demonstrates the capacity to reduce severe pulmonary hypertension, right ventricular hypertrophy and induce apoptosis of hyperproliferative cells in pre-capillary pulmonary arterioles.

The substance P fragment SP(1-7) stimulates motor behavior and nigral dopamine release.

Earlier studies have shown that the undecapeptide substance P (SP) alters motor behavior and dopamine metabolism following injection into the substantia nigra (SN) in rat, even though the SN appears largely devoid of SP-specific (NK-1) receptors. In this report, intra-nigral injections of the amino-terminal SP fragment SP(1-7) enhanced rearing, sniffing and locomotor activity, and increased the nigral DOPAC-to-DA ratio. In addition, SP(1-7) increased 3H-DA release from the SN in vitro. These findings suggest that some of the effects of nigral SP on motor behavior and dopamine release are mediated by amino-terminal fragments of SP.

Combination cancer chemotherapy with one compound: Pluripotent bradykinin antagonists

Lung and prostate cancers are major health problems worldwide. Treatments with standard chemotherapy agents are relatively ineffective. Combination chemotherapy gives better treatment than a single agent because the drugs can inhibit the cancer in different pathways, but new therapeutic agents are needed for the treatment of both tumor types. Bradykinin (BK) antagonists offer advantages of combination therapy in one compound. These promising multitargeted anti-cancer compounds selectively stimulate apoptosis in cancers and also inhibit both angiogenesis and matrix metalloprotease (MMP) action in treated lung and prostate tumors in nude mice. The highly potent, metabolism-resistant bradykinin antagonist peptide dimer, B-9870 [SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg)2] (SUIM=suberimidyl; Hyp=4-hydroxyproline; Igl=alpha-(2-indanyl)glycine; Oic=octahydroindole-2-carboxylic acid) and its non-peptide mimetic, BKM-570 [2,3,4,5,6-pentafluorocinnamoyl-(o-2,6-dichlorobenzyl)-L-tyrosine-N-(4-amino-2,2,6,6-tetramethylpiperidyl)amide] are superior to the widely used but toxic chemotherapeutic drugs cisplatin and taxotere. In certain combinations, they act synergistically with standard anti-cancer drugs. Due to its structure and biological activity, BKM-570 is an attractive lead compound for derivatization and evaluation for lung and prostate cancer drugs.

Modulation of blood pressure by substance P: opposite effects of N- and C-terminal fragments on anesthetized rats

Considerable evidence suggests that substance P (SP) may be a transmitter mediating the depressor effects of baroreceptor reflex activation within the brainstem, yet intracerebroventricular administration of SP is reported to result in a pronounced pressor effect. In this study, SP injected into the 4th cerebral ventricle produced a biphasic effect; a brief decrease in blood pressure followed by a lengthy increase. Similar injections of a carboxy-terminal fragment of SP produced only a pressor effect of long duration. Injection of an amino-terminal SP fragment produced only a brief, rapid depressor effect. These results suggest that the amino-terminal sequence of SP may be involved in mediating the depressor effects of baroreceptor activation.

Non‐competitive pharmacological antagonism at the rabbit B1 receptor

The B1 receptor for kinins, stimulated by kinin metabolites without the C‐terminal Arg residue (e.g., des‐Arg9‐bradykinin (BK) and Lys‐des‐Arg9‐BK), is an increasingly recognized molecular target for the development of analgesic and anti‐inflammatory drugs. Recently developed antagonists of this receptor were compared to a conventional antagonist, Ac‐Lys‐[Leu8]‐des‐Arg9‐BK, in pharmacological assays based on the rabbit B1 receptor. B‐9858 (Lys‐Lys‐[Hyp3, Igl5, D‐Igl7, Oic8]des‐Arg9‐BK) and three other analogues possessing the α‐2‐indanylglycine5 (Igl5) residue (order of potency B‐9858 ∼ B‐10146>B‐10148>B‐10050) were partially insurmountable antagonists of des‐Arg9‐BK in the contractility assay based on rabbit aortic rings. B‐9858‐induced depression of the maximal effect was more pronounced in tissues treated with the protein synthesis inhibitor cycloheximide to block the spontaneous increase of response attributed to the post‐isolation formation of B1 receptors, and only partly reversible on washing. By comparison, Ac‐Lys‐[Leu8]des‐Arg9‐BK was a surmountable antagonist (pA2 7.5), even in cycloheximide‐treated tissues. B‐9958 (Lys‐[Hyp3, CpG5, D‐Tic7, CpG8]des‐Arg9‐BK) was also surmountable (pA2 8.5). The binding of [3H]‐Lys‐des‐Arg9‐BK to recombinant rabbit B1 receptors expressed in COS‐1 cells was influenced by two of the antagonists: while Ac‐Lys‐[Leu8]des‐Arg9‐BK competed for the radioligand binding without affecting the Bmax, B‐9858 decreased the Bmax in a time‐dependent and washout‐resistant manner. B‐9858 and analogues possessing Igl5 are the first reported non‐competitive, non‐equilibrium antagonists of the kinin B1 receptor.

Small zone gel chromatography of interacting systems: Theoretical and experimental evaluation of elution profiles for kinetically controlled macromolecule-ligand reactions

A phenomenological theory of small zone gel chromatography is elaborated for kinetically controlled macromolecule-ligand interactions. Chromatography, direct binding experiments, and rate measurements are used for successful comparison of experimental behavior with theoretical elution profiles for the interaction of MAP-2 with two synthetic peptide fragments from the C-terminal moieties of alpha- and beta-tubulin subunits. The results of this study provide guidelines for interpretation of experimental small zone elution profiles of total ligand.

Structural modification of the highly potent peptide bradykinin B1 receptor antagonist B9958

Bradykinin (BK)-related peptides stimulate two major classes of receptors, B1 and B2. The B1 receptor (B1R) plays an important role in various pathophysiological states including chronic inflammation, pain, hypotension, trauma and proliferation of cancer. Therefore, there is interest in the development of highly potent peptide BK B1R antagonists. We previously developed a highly potent and selective BK B1R receptor antagonist, B9958 (Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK) (Hyp, trans-4-hydroxyproline; CpG, alpha-cyclopentylglycine; Tic, tetrahydroisoquinoline-3-carboxylic acid). We now report on new BK B1R antagonist analogs of B9958 with N-terminal basic residues in the d-configuration, or Lys-, Orn- derivatives (NiK, epsilon-nicotinoyllysine; PzO, 3-pyrazinoylornithine) and/or having hindered unusual amino acids at position 5 (Igl, alpha-(2-indanyl)glycine). These changes were designed to prevent enzyme degradation while keeping an acceptable affinity. However, these new analogs do not show higher B1R antagonist activity than B9958, but its N-terminal acylated derivative with a bulky and hydrophobic 2,3,4,5,6-pentafluorocinnamic acid (F5c), B10324, retains a B1R antagonist activity close to that of B9958 and, in addition, has high inhibition in vivo against lung cancer (SCLC, 86 %) and moderate inhibition against prostate cancer (PC3, 43%) xenografts. This class of compounds offers hope for the development of new BK antagonist peptide drugs for lung or prostate cancer.

Antagonist, partial agonist and antiproliferative actions of B‐9870 (CU201) as a function of the expression and density of the bradykinin B1 and B2 receptors

A bradykinin (BK) B2 receptor (B2R) antagonist, B‐9870 (CU201), has been proposed to behave as a ‘biased agonist’ at B2Rs and to exert anti‐neoplasic effects. It was unclear whether these effects were determined by the activation of B2Rs by the drug. B‐9870 was evaluated for antagonism or stimulation of several responses mediated by the rabbit B2R or B1 receptor (B1R); its anti‐proliferative activity was also characterized.