Chapter 2. Historical.

2.1. Introduction.

This review reproduces the latest advances in the design of carnitine acyltransferase inhibitors, and provides only the background necessary for a fruitful discussion of the conformational behavior of the inhibitors mentioned below.

2.2. Acyltransferase inhibitors.

One common strategy to design enzymatic inhibitors is to use transition-structure-analog theory [Wolfenden, 1969] to design a compound that the enzyme can recognize. Binding of the following tetrahedral intermediate is commonly considered important in the transfer of acyl groups to CoA:

Figure 2.1. Tetrahedral intermediate for Carnitine-CoA acyl transfer. [fig.gandour93.gif, 1.231KB ]

We can classify enzymatic inhibitors according to their observed effects on enzymatic activity (reversible or irreversible) or by their mechanism of action (competitive or noncompetitive). Irreversible inhibitors eliminate enzymatic activity in such a way that the enzyme does not recover its original activity. Reversible inhibitors allow the recovery of catalytic activity after elimination of the inhibitor. Competitive inhibitors bind to the active site instead of the substrate, while noncompetitive inhibitors do not attach themselves to the active site, so the substrate can still bind to the enzyme. Thus we have four main types of enzymatic inhibitors:

1. Competitive reversible
2. Competitive irreversible
3. Noncompetitive reversible
4. Noncompetitive irreversible

2.2.1. Competitive reversible inhibitors.

(S)-Carnitine and (S)-acetylcarnitine inhibit competitively pigeon CAT. [Tipton, 1969] Tipton and Chase justified the recognition of the S isomers by postulating a two-point recognition site that binds only to carboxylate and ammonium. CATs extracted from bovine heart, pig heart and mouse-liver peroxisomes discriminated substantially between the (R) and the (S) isomers. The authors postulated a more restricted two-point recognition site, or a three-point recognition site for these CATs.

Aminocarnitine

Figure 2.2. Aminocarnitine.[fig.aminocarn.gif, 0.595KB.]

is a weak reversible competitive inhibitor for CAT, although it inhibits strongly both CPT-I and CPT-II. [Kanamaru, 1985] [Jenkins, 1985] [Jenkins, 1986] Due to the isosteric relationship to carnitine, N-Acylated derivatives of racemic aminocarnitine can also inhibit reversibly carnitine acyltransferases, as shown by the great inhibitory potency of D,L-acetylaminocarnitine. [Jenkins, 1985] (R)-acetylaminocarnitine --named ``emeriamine'' because it was isolated from a culture filtrate of Emericella quadrilineata-- showed even stronger inhibitory action than the racemic compound. [Shinagawa, 1987]

Gandour et al. [Gandour, 1986] [Gandour, 1992] showed good inhibition of CAT by hemiacylcarnitinium (HAC). Later, the same authors [Gandour, 1988] [Gandour, 1993] demonstrated the strong activity of hemipalmitoylcarnitinium (HPC) as a competitive inhibitor of CPT-I. HAC inhibits CAT, but not CPT. HPC inhibits CPT, but not CAT. It seemed that CPT requires a long chain for proper recognition of the substrate. These differences in the specificity of the inhibitors allowed for a partial mapping of the topographies of the active sites in both enzymes. The design of all these inhibitors relied on making them analogs of the [tetrahedral intermediate] mentioned previously by means of restricting the conformations with a morpholinium moiety:

Figure 2.3. Hemiacylcarnitinium moiety. [fig.HAC.gif, 0.740KB ]

The morpholinium ring locks the N-CH₂-CH-OH(Ac) torsion of the carnitine backbone in the gauche conformation. The authors stated the difficulty of excluding selective binding of the open form of the hemiacetal, but "presumably" hemiacylcarnitiniums bind as cyclic structures. NMR did not detect any open form of the compound in solution.

Saeed et al. [Saeed, 1993] showed that 3-hydroxy-5,5-dimethylhexanoic acid

Figure 2.4. 3-hydroxy-5,5-dimethylhexanoic acid. [fig.saeed93.gif, 0.581KB ]

competitively inhibited CAT, CPT-I and CPT-II, indicating that these enzymes require a positively charged center to show catalysis. Saeed et al. [Saeed, 1994] later reported the inhibitory properties of 3-amino-5,5-dimethylhexanoic acid.

Figure 2.5. 3-amino-5,5-dimethylhexanoic acid.[fig.saeed94.gif, 0.585KB ]

Both 3-hydroxy- and 3-amino-5,5-dimethylhexanoic acid are alternative substrates for CAT.

Gandour's group evaluated Protein Kinase C, COT and CPT inhibitors based on a 2-oxo-1,3,6-dioxazaphosphacinium moiety. [Kumaravel, 1994]

Figure 2.6. 4-hexadecyl-2,4,4-trimethyl-2-oxo-1,3,6-dioxazaphosphacinium bromide.[fig.kumaravel94.gif, 0.743KB ]

The authors tested racemic mixtures (2S, 4S)/(2R, 4R) and (2S, 4R)/(2R, 4S) and found they inhibited COT and CPT-II moderately.

Gandour's group also reported several CAT inhibitors incorporating a methylenecarboxylate substructure on a morpholinium ring. [Sun, 1995]

Figure 2.7. 6-(Carboxylatomethyl)-2-(hydroxymethyl)-2,4,4-trimethylmorpholinium.[fig.sun95.gif, 1.157KB ]

All four stereoisomers were evaluated for inhibitory activity and the authors concluded that ``CAT recognizes both configurations at C2 and C6 in the analogues.''

Anderson et al. [Anderson, 1995] reported on the potency of a series of CPT-I inhibitors based on a phosphonate moiety designed by transition-structure-analog theory.

Figure 2.8. Anderson's phosphonate.[fig.anderson951.gif, 0.842KB ]

This compound showed appreciable inhibitory activity. After trying systematic structural variations, they found that the following phosphate:

Figure 2.9. Anderson's phosphate.[fig.anderson952.gif, 0.980KB ]

inhibited to a level comparable to the phosphonate. Both the phosphonate and the phosphate could show similar detergency, thus explaining the similar inhibition. The authors discarded such an explanation due to the stronger inhibition shown by the R enantiomers when compared to the S enantiomers.

2.2.2. Competitive irreversible inhibitors.

We can visualize the enzyme and the substrates as part of a ternary complex. Bisubstrate inhibitors resemble the spatial disposition of the substrates, leaving the enzyme as the "third" component. One of the earliest examples of a bisubstrate inhibitor is S-carboxymethyl-CoA-(R)-carnitine ester, reported by Chase and Tubbs. Bromoacetyl-CoA and bromoacetyl-(R)-carnitine are also bisubstrate inhibitors for CAT. [Chase, 1969]

Fritz and Schultz [Fritz, 1965] reported inhibition of CAT by p-hydroxymercuribenzoate (HMB) at a concentration of 2.6 {micro}M. Such inhibition was prevented or partially reversed by addition of acetyl-CoA, which suggested to these authors that HMB may bind to the active site.

Several groups have reported compounds possessing inhibitory activity towards carnitine acyltransferases. Etomoxir (2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate) is an irreversible inhibitor of CPT-I. [Eistetter, 1982] Tetradecylglycidate (TDGA) is also an oxirane-based irreversible competitive inhibitor of CPT-I. [Tutwiler, 1985]

Another competitive inhibitor is methoxycarbonylCoA disulfide, [Venkatraghavan, 1983] which modifies selectively a sulfhydryl group in the active site. Dithioerythritol or thiocholine can partially reverse its action.

2.2.3. Noncompetitive reversible inhibitors.

5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), reacts with sulfhydryl groups in a non-specific way. It inhibits pig heart CAT [Fritz, 1963] and pigeon breast CAT. [Venkatraghavan, 1983] Because this reagent forms disulfide linkages, adding a disulfide exchange reagent such as dithiothreitol, dithioerythritol or thiocholine can restore enzymatic activity.

Derrick and Ramsay [Derrick, 1989] showed that malonylCoA is a reversible inhibitor of peroxisomal CPT-I, but does not show competitive behavior. These authors estimated that peroxisomal palmitoyltransferase activity constituted up to 20% of the peroxisomal + overt-mitochondrial pool in fed-rat liver. A concentration lower than 10{micro}M of malonyl-CoA was enough to show 90% inhibition of the peroxisomal palmitoyltranferase.

2.2.4. Noncompetitive irreversible inhibitors.

Fritz and Schultz [Fritz, 1969] reported the inhibitory activity of ZnCl₂, HgCl₂, N-ethylmaleimide and iodoacetamide. Addition of ethanethiol prevented or partially reversed HgCl₂ inhibition. The authors did not cite the values of K_i for these compounds.