Hydroxynaphthaldehyde Phosphate Derivatives as Potent Covalent Schiff Base Inhibitors of Fructose-1,6-bisphosphate Aldolase

Abstract

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design.

Anticancer Agents That Counteract Tumor Glycolysis

“…Similarly to salicylic derivative 44 , the presence of the hydroxyl group ortho to the aldehyde in 46 proved to be essential in determining its mechanism of action (Schiff base formation). [161] Hexitol diphosphate, consisting in a diastereoisomeric mixture of glucitol bisphosphate ( 47 ) and mannitol bisphosphate ( 48 ), had been known a long time as a competitive inhibitor of class I muscle ALD ( K i ~1.2 μM). [162] More recently 47 and 48 were separately tested on rabbit muscle aldolase, and they both behaved as competitive inhibitors with K i values of 100 μM ( 47 ) and 7.3 μM ( 48 ).…”

Multiple Pathways for the Irreversible Inhibition of Steroid Sulfatase with Quinone Methide‐Generating Suicide Inhibitors

“…This species, whose presence was confirmed by spiking the STS-catalyzed reaction with an authentic sample of independently synthesized 4-diFME1 [3] (Figure 3 F), is formed as an unstable intermediate and rapidly decomposes to give 4-FE1 ( Figure 3 F and G). Because aldehydes have been known to act as highly potent and sometimes almost irreversible enzyme inhibitors, [10] we reasoned that inactivation of STS by compound 4 might be mainly due to inhibition by 4-FE1. Indeed, incubation of STS with just 4-FE1 resulted in time and concentration-dependent inhibition of STS (Figure 4 A).…”

“…[13] Although the site of STS modification by 4-FE1 has yet to be established, it is highly likely that it is forming a Schiff base with an active-site residue because it has been shown that certain aldehydes can function as almost irreversible enzyme inhibitors through the formation of stable Schiff base adducts with residues bearing side-chain amines. [10] Lys368 in the active site of STS is a strong candidate for Schiff base formation A C H T U N G T R E N N U N G because it is in close proximity to the 4-position of estrone, which has been modeled into the active site of STS. [14] It is possible that the formyl group of 2-FE1 cannot attain the correct geometry or is too far removed from Lys368 to form a stable Schiff base.…”

Cloning, expression, and partial characterization of FBPA from Schistosoma japonicum, a molecule on that the fluke may develop nutrition competition and immune evasion from human

“…Schistosome cells express FBPA that upon degradation give rise to similar antigenic peptides that are presented on the cell surface complexed with MHC class I molecules (MHC I), that greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes (Apcher et al 2010;Zervoudi et al 2013), and as a result cannot develops cytotoxic cellular responses contributing to immune evasion by schistosome cells. Unlike the other molecules of S. japonicum, such as protein arginine methyltransferase 1 and allergen-like protein 1, which could induce mouse bone marrow-derived dendritic cell (BMDC) to mature and therefore activate their immune response (Diao et al 2014), or induce a polarized Th2 immune response by increasing specific antibody significantly in S. japonicum-infected mice (Chen et al 2010), sjFBPA contains similar antigenic peptides with that of human FBPAs and may contribute to immune evasion of S. japonicum from their host, human being. This result would be confirmed by some animal experiment.…”