Allosteric modulation of semicarbazide‐sensitive amine oxidase activities in vitro by imidazoline receptor ligands

Abstract

1 Evidence indicates that imidazoline I 2 binding sites (I 2 BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. 2 We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. 3 While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and b-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. Abbreviations: a, factor by which K M is altered by imidazoline drug; AM, amiloride; b, factor by which V max is altered by imidazoline drug; 2-BFI, 2-(2-benzofuranyl)-2-imidazoline; BPAO, bovine plasma amine oxidase; BZ, benzylamine; CIR, cirazoline; D, imidazoline drug binding to any site on BPAO; E, enzyme; E max , maximum degree to which measured parameter can be changed by drug of interest; g, factor by which affinity of enzyme for substrate is reduced by a mixed inhibitor; H, imidazoline drug binding to high-affinity site on BPAO; I, imidazoline drug binding to (very low-affinity) inhibitory site on BPAO; IBS, imidazoline binding site; IDZ, idazoxan; I 1 R, imidazoline type-1 receptor; k p , rate constant for enzymatic formation of product; K H , dissociation constant for ligand H from high-affinity site on BPAO; K L , dissociation constant for ligand L from low-affinity site on BPAO; K S , dissociation constant for substrate S from active site of BPAO; L, imidazoline drug binding to low-affinity site on BPAO; MA, methylamine; MOX, moxonidine; P, product (of an enzyme reaction

Multiple Binding Sites for Substrates and Modulators of Semicarbazide-Sensitive Amine Oxidases: Kinetic Consequences

“…However, the ping-pong nature of substrate oxidation by hSSAO and BPAO raises the possibility that substrates may bind to both oxidized and reduced enzyme forms, yielding hormetic kinetic plots, and recent kinetic analyses provide indirect evidence consistent with this possibility (Holt et al, 2007). Data from our laboratory also reveal an ability of several imidazoline binding site ligands to cause partial inhibition, or activation, of BPAO activity (Holt et al, 2004). Kinetic modeling implicated the involvement of up to four distinct sites through which these ligands exert their effects, although neither the locations of the putative sites nor their functional interrelationships could be deduced from the models.…”

“…Previous kinetic analyses indicated that CLON and other imidazoline ligands bind to four sites on BPAO (Holt et al, 2004). Therefore, we hypothesized that two of these sites correspond to the active site on an oxidized and reduced the TPQ into an inactive on-copper conformation.…”

“…1. For BPAO, they were TPP ϩ (Di Paolo et al, 2004), clonidine (CLON) (Holt et al, 2004), guanabenz (GBZ) (Ozaita et al, 1997;Holt et al, 2004), (ϩ)-tranylcypromine [(ϩ)TCP)] (Carpéné et al, 1995), and 2-(2-benzofuranyl)-2-imidazoline (2-BFI) (Holt et al, 2004); and for hSSAO, they were (ϩ)TCP, GBZ, and (ϩ)-amphetamine [(ϩ)AM] (Lyles, 1984). Although fluorescence quenching or inhibition of peroxidase have been observed with several amine oxidase substrates and inhibitors (Sayre et al, 1996;Holt and Palcic, 2006), preliminary screening assays confirmed that none of the modulators used interfered with chromogenic or fluorogenic reagents.…”

Imidazoline Antihypertensive Drugs: Selective I1‐Imidazoline Receptors Activation

“…The I 2 receptors are allosteric binding site associated with the catalytic site of monoamine oxidase (MAO), but also on other non‐MAO oxidative enzymes [22].…”

Guinea-pig ileum as ex vivo model useful to characterize ligands displaying Imidazoline I2 and Adrenergic alpha2 mixed activity: a preliminary study

“…5 I2-IB subtype, originally described as the ImidazolineGuanidinium Receptive Site (IGRS) and characterized by idazoxan binding, 6 has been identified as allosteric binding site on monoamine oxidase (MAO) and on other non-MAO oxidative enzymes. 7 The interest about this I2 subtype is due to its involvement in neuropathic and inflammatory pain. 8 Neuropathic pain is a condition often refractory to the classical pharmacological approach [opioids and nonsteroidal anti-inflammatory drugs (NSAIDs)] that is treated by tricyclic antidepressants (TCAs), anticonvulsants and systemic local anesthetics.…”