Novel Screen Methodologies for Identification of New Microbial Metabolites
105
pyripyropene A was found to be reversible and non-competitive with the
substrate oleoyl-CoA; it appeared to be competitive with cholesterol which
pyripyropene A structurally resembles [143]. Around 150 derivatives of pyripyropene A have been synthesised to investigate the structural features of the
molecule responsible for ACAT activity [143]. One of these, a 1,11-benzylidene
acetal-7-O-n-valeryl derivative, PR109, was thirteen times more potent than
pyripyropene A in vitro and more effective in vivo. The 13-hydroxy group and
pyridine moiety were found to be very important for activity.
The terpendoles from Albophoma yamanashiensis constitute a series of epoxidated indoloditerpene derivatives [ 144, 145]. Terpendole D (structure shown
in Fig. 8) was stated to be the most specific microbial ACAT inhibitory yet
evaluated in the J774 macrophage assay, as judged by its ratio of ACAT
inhibitory activity to cytotoxicity.
AS-183 is a novel furan-3-one derivative which exhibits potent inhibition in
the in vitro ACAT enzyme assay [146]. It had no effect on acyl CoA:
sn-glycerol-3-phosphate acyltransferase (AGAT), lecithin: cholesterol acyltransferase (LCAT), cholesterol 7cx-hydroxylase, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. The compound did affect cholesterol
ester formation in vitro in HepG2 (human liver), CaCO 2 (human small intestine)
and THP-1 (human monocyte) cell lines.
Lateritin, a substituted 1,4-perhydrooxazine-2,5-dione derivative from
Gibberella lateritium [147] and gypsetin, a complex diketopiperazine from
Nannizzia gypsea vat. incurvata [148, 149], inhibited cholesteryl ester formation
in J774 macrophages and did not affect surface binding, uptake or degradation
of oxidised [lzsI]-LDL.
Squalene synthase catalyses the first pathway specific step in cholesterol
biosynthesis, and inhibitors of this enzyme may have a cholesterol-lowering
effect.
Screening of microbial samples using a squalene synthase assay involving rat
liver homogenate protein, [1-1~C]-isopentenyl pyrophosphate (IPP) and trans,
trans-[2-14C]-farnesyl pyrophosphate led to the discovery of the squalestatins.
This assay allows measurement of squalene production from either [1-14C]-IPP
as a radiolabelled precursor of FPP, or from [2-14C]-FPP as a direct substrate
for squalene synthase. Ascorbate and ascorbate oxidase were included in the
reaction mixture to consume oxygen and prevent further conversion of IPP or
FPP to metabolites beyond squalene. The squalestatins were discovered as
potent inhibitors of squalene production from [1-14C]-IPP or [2-14C]-FPP
[150,151].
Squalestatins S1-3 are Phoma sp. metabolites containing a trihydroxylated,
tricarboxylated 2,8-dioxobicyclo-[3.2.1]octane core. Squalestatin 1 (the structure of which is shown in Fig. 8) is identical to zaragozic acid A, which was
isolated from an unidentified sterile fungal culture as a result of its squalene
synthase inhibitory activity [152]. The squalestatins were shown to be potent
inhibitors of rat liver squalene synthase activity, and squalestatin 1 was a potent
inhibitor of Candida albicans microsomal squalene synthase. Zaragozic acid
105
pyripyropene A was found to be reversible and non-competitive with the
substrate oleoyl-CoA; it appeared to be competitive with cholesterol which
pyripyropene A structurally resembles [143]. Around 150 derivatives of pyripyropene A have been synthesised to investigate the structural features of the
molecule responsible for ACAT activity [143]. One of these, a 1,11-benzylidene
acetal-7-O-n-valeryl derivative, PR109, was thirteen times more potent than
pyripyropene A in vitro and more effective in vivo. The 13-hydroxy group and
pyridine moiety were found to be very important for activity.
The terpendoles from Albophoma yamanashiensis constitute a series of epoxidated indoloditerpene derivatives [ 144, 145]. Terpendole D (structure shown
in Fig. 8) was stated to be the most specific microbial ACAT inhibitory yet
evaluated in the J774 macrophage assay, as judged by its ratio of ACAT
inhibitory activity to cytotoxicity.
AS-183 is a novel furan-3-one derivative which exhibits potent inhibition in
the in vitro ACAT enzyme assay [146]. It had no effect on acyl CoA:
sn-glycerol-3-phosphate acyltransferase (AGAT), lecithin: cholesterol acyltransferase (LCAT), cholesterol 7cx-hydroxylase, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. The compound did affect cholesterol
ester formation in vitro in HepG2 (human liver), CaCO 2 (human small intestine)
and THP-1 (human monocyte) cell lines.
Lateritin, a substituted 1,4-perhydrooxazine-2,5-dione derivative from
Gibberella lateritium [147] and gypsetin, a complex diketopiperazine from
Nannizzia gypsea vat. incurvata [148, 149], inhibited cholesteryl ester formation
in J774 macrophages and did not affect surface binding, uptake or degradation
of oxidised [lzsI]-LDL.
Squalene synthase catalyses the first pathway specific step in cholesterol
biosynthesis, and inhibitors of this enzyme may have a cholesterol-lowering
effect.
Screening of microbial samples using a squalene synthase assay involving rat
liver homogenate protein, [1-1~C]-isopentenyl pyrophosphate (IPP) and trans,
trans-[2-14C]-farnesyl pyrophosphate led to the discovery of the squalestatins.
This assay allows measurement of squalene production from either [1-14C]-IPP
as a radiolabelled precursor of FPP, or from [2-14C]-FPP as a direct substrate
for squalene synthase. Ascorbate and ascorbate oxidase were included in the
reaction mixture to consume oxygen and prevent further conversion of IPP or
FPP to metabolites beyond squalene. The squalestatins were discovered as
potent inhibitors of squalene production from [1-14C]-IPP or [2-14C]-FPP
[150,151].
Squalestatins S1-3 are Phoma sp. metabolites containing a trihydroxylated,
tricarboxylated 2,8-dioxobicyclo-[3.2.1]octane core. Squalestatin 1 (the structure of which is shown in Fig. 8) is identical to zaragozic acid A, which was
isolated from an unidentified sterile fungal culture as a result of its squalene
synthase inhibitory activity [152]. The squalestatins were shown to be potent
inhibitors of rat liver squalene synthase activity, and squalestatin 1 was a potent
inhibitor of Candida albicans microsomal squalene synthase. Zaragozic acid