Synthesis and structure-activity relations of bestatin analogs, inhibitors of aminopeptidase B

Nishizawa, Rinzo; Saino, Tetsushi; Takita, Tomohisa; Suda, Hiroyuki; Aoyagi, Takaaki; Umezawa, Hamao

doi:10.1021/jm00214a010

Cited by 162 publications

(58 citation statements)

References 3 publications

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“…The a-amino group of bestatin, which has a pKa of 8.1 (16), is probably the NH2 form when coordinated to the positively charged zinc ion at physiologic pH. The (S)-hydroxyl group of bestatin is also coordinated to the readily exchangeable zinc ion, and, therefore, the mode of binding of the a-amino-f3-hydroxyl moiety resembles that proposed by Nishizawa et al (17) and not that of Nishino and Powers (18) …”

Section: Requirements For Lap-catalyzed Peptide Hydrolysismentioning

confidence: 52%

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

Burley

David

Lipscomb

1991

Proc. Natl. Acad. Sci. U.S.A.

146

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The three-dimensional structures of native bovine lens leucine aminopeptidase (EC 3.4.11.1) and its complex with bestatin, a slow-binding inhibitor, have been solved and exhaustively refined. The mode of binding of bestatin to leucine aminopeptidase may be similar to that of a tetrahedral intermediate that is thought to form during peptide bond hydrolysis. Bestatin binds in the active site with its a-amino group and hydroxyl group coordinated to the zinc ion located in the readily exchangeable divalent cation binding site. Its phenylalanyl side chain is stabilized by van der Waals interactions with Met-270, Thr-359, Gly-362, Aa-451, and Met-454, which appear to form a terminal hydrophobic pocket. The leucyl side chain binds in another hydrophobic cleft lined by Asn-330, Ala-333, and fle-421. Hydrogen bonds involving active site residues Lys-262, Asp-273, Gly-360, and Leu-362 are responsible for stabilizing the backbone nitrogen and oxygen atoms of bestatin. The mode of bestatin inhibition of leucine aminopeptidase is discussed and correlated with biochemical studies of bestatin analogues. In addition, features of a mechanism of catalysis of peptide hydrolysis by leucine aminopeptidase are discussed.Leucine aminopeptidase [LAP; cytosol aminopeptidase, a-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1] is a widely distributed cytosolic exopeptidase-which catalyzes the hydrolysis of amino acids from the amino terminus of polypeptide chains (for reviews, see refs. 1-3). As their name implies, the LAPs cleave leucyl substrates, although substantial rates of enzymatic cleavage are seen with most amino-terminal amino acids. However, residues P1 or PI should not be lysine or arginine, P1 should not be a D-amino acid, and P' should not be hydroxyproline or proline, using the convention of Schechter and Berger (4). The amino acid occurring at P' does not seem to influence the rate ofcatalysis significantly (5). LAP also catalyzes the hydrolysis of amino acid amides, alkylamides, arylamides, and hydrazides and has some esterase activity.Bovine lens LAP is a hexameric enzyme of molecular weight 324,000, which consists of six identical subunits (6) of molecular weight 54,000 and 12 zinc ions (7). A potent, slow-binding inhibitor of LAP, bestatin or [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine hydroxide, was isolated from culture filtrates of Streptomyces olivoreticuli (8) and was shown to have a Ki = 20 nM for bovine lens LAP (9).The three-dimensional structure of the bestatin-inhibited enzyme has been solved in our laboratory at 3-A resolution by x-ray crystallography using the multiple isomorphous replacement method with phase combination and density modification (10). Recently, we reported the refinement of the structures of both the native enzyme and the LAPbestatin complex at 2.32-and 2.25-A resolution, respectively (11). In this paper, we present a detailed analysis of the mechanism of inhibition of the enzyme by bestatin and discuss features of a mechanism of catalysis of peptide hydrolysis by LAP...

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Section: Requirements For Lap-catalyzed Peptide Hydrolysismentioning

confidence: 52%

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

Burley

David

Lipscomb

1991

Proc. Natl. Acad. Sci. U.S.A.

146

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“…We decided to use the natural product, bestatin, as the scaffold for the development of ABPs for MAPs because it is a general MAP inhibitor, kills Plasmodium parasites, and is synthetically tractable using both solution and solid-phase chemistry (22,35,36). To identify the target(s) of bestatin in P. falciparum, we utilized a previously published bestatin-based ABP, MH01 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Harbut

Velmourougane

Dalal

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

133

176

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Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.protease | chemical-genetics | proteomics | small molecule | drug design

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“…The peptide backbone of the compounds was stepwise assembled by classical methods, using Boc as the ␣-amino protecting group and benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate as the coupling reagent, either in homogeneous phase or on solid phase with methylbenzhydrylamine resin, necessitating a final HF cleavage procedure. Published protocols were followed for the formation of the peptide bond isostere moieties, reduced amide bond (Leu-⌿(CH s NH)-Asp in JMV963) (21,22), norstatine (Norsta-containing compounds) (23,24), and statine and analogs (Sta-, AHPPA-, ACHPA-containing compounds) (25,26). All synthetic inhibitors were purified on C18 reverse-phase HPLC, and their purity and identity were assessed by reverse-phase HPLC and electrospray mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

BACE1- and BACE2-expressing Human Cells

Andrau

Dumanchin-Njock

Ayral

et al. 2003

Journal of Biological Chemistry

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We have set up stably transfected HEK293 cells overexpressing the ␤-secretases BACE1 and BACE2 either alone or in combination with wild-type ␤-amyloid precursor protein (␤APP). The characterization of the ␤APP-derived catabolites indicates that cells expressing BACEs produce less genuine A␤1-40/42 but higher amounts of secreted sAPP␤ and N-terminal-truncated A␤ species. This was accompanied by a concomitant modulation of the C-terminal counterpart products C89 and C79 for BACE1 and BACE2, respectively. These cells were used to set up a novel BACE assay based on two quenched fluorimetric substrates mimicking the wild-type (JMV2235) and Swedish-mutated (JMV2236) ␤APP sequences targeted by BACE activities. We show that BACEs activities are enhanced by the Swedish mutation and maximal at pH 4.5. The specificity of this double assay for genuine ␤-secretase activity was demonstrated by means of cathepsin D, a "false positive" BACE candidate. Thus, cathepsin D was unable to cleave preferentially the JMV2236-mutated substrate. The selectivity of the assay was also emphasized by the lack of JMV cleavage triggered by other "secretases" candidates such as ADAM10 (A disintegrin and metalloprotease 10), tumor necrosis ␣-converting enzyme, and presenilins 1 and 2. Finally, the assay was used to screen for putative in vitro BACE inhibitors. We identified a series of statine-derived sequences that dose-dependently inhibited BACE1 and BACE2 activities with IC 50 in the micromolar range, some of which displaying selectivity for either BACE1 or BACE2.

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Synthesis and structure-activity relations of bestatin analogs, inhibitors of aminopeptidase B

Cited by 162 publications

References 3 publications

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

BACE1- and BACE2-expressing Human Cells

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