ABSTRACT
Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans.
Subject(s)
Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans/drug effects , Cyclophilin A/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Cyclophilin A/metabolism , Cyclosporine , Drug Design , Ligands , Models, Molecular , Protein Binding , Spectrometry, Mass, Electrospray IonizationABSTRACT
We present the first study of protein regulation by ligands in Caenorhabditis elegans. The ligands were peptidyl-prolyl isomerase inhibitors of cyclophilins. Up-regulation is observed for several heat shock proteins and one ligand in particular caused a greater than 2-fold enhancement of cyclophilin CYN-5. Additionally, several metabolic enzymes display elevated levels. This approach, using label-free relative quantification, provides an extremely attractive way of measuring the effect of ligands on an entire proteome, with minimal sample pretreatment, which could be applicable to large-scale studies. In this initial study, which compares the effect of three ligands, 54 unique proteins have been identified that are up- (51) or down- (3) regulated in the presence of a given ligand. A total of 431 C. elegans proteins were identified. Our methodology provides an intriguing new direction for in vivo screening of the effects of novel and untested ligands at the whole organism level.
Subject(s)
Caenorhabditis elegans/chemistry , Cyclophilins/drug effects , Gene Expression Regulation/drug effects , Proteome/drug effects , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cyclophilins/genetics , Ligands , Peptidylprolyl Isomerase/pharmacologyABSTRACT
Monoamine oxidase from Aspergillus niger (MAO-N) is a flavoenzyme that catalyses the oxidative deamination of primary amines. MAO-N has been used as the starting model for a series of directed evolution experiments, resulting in mutants of improved activity and broader substrate specificity, suitable for application in the preparative deracemisation of primary, secondary and tertiary amines when used as part of a chemoenzymatic oxidation-reduction cycle. The structures of a three-point mutant (Asn336Ser/Met348Lys/Ile246Met or MAO-N-D3) and a five-point mutant (Asn336Ser/Met348Lys/Ile246Met/Thr384Asn/Asp385Ser or MAO-N-D5) have been obtained using a multiple-wavelength anomalous diffraction experiment on a selenomethionine derivative of the truncated MAO-N-D5 enzyme. MAO-N exists as a homotetramer with a large channel at its centre and shares some structural features with human MAO B (MAO-B). A hydrophobic cavity extends from the protein surface to the active site, where a non-covalently bound flavin adenine dinucleotide (FAD) sits at the base of an 'aromatic cage,' the sides of which are formed by Trp430 and Phe466. A molecule of l-proline was observed near the FAD, and this ligand superimposed well with isatin, a reversible inhibitor of MAO-B, when the structures of MAO-N proline and MAO-B-isatin were overlaid. Of the mutations that confer the ability to catalyse the oxidation of secondary amines in MAO-N-D3, Asn336Ser reduces steric bulk behind Trp430 of the aromatic cage and Ile246Met confers greater flexibility within the substrate binding site. The two additional mutations, Thr384Asn and Asp385Ser, that occur in the MAO-N-D5 variant, which is able to oxidise tertiary amines, appear to influence the active-site environment remotely through changes in tertiary structure that perturb the side chain of Phe382, again altering the steric and electronic character of the active site near FAD. The possible implications of the change in steric and electronic environment caused by relevant mutations are discussed with respect to the improved catalytic efficiency of the MAO-N variants described in the literature.
Subject(s)
Aspergillus niger/enzymology , Directed Molecular Evolution , Monoamine Oxidase/chemistry , Monoamine Oxidase/metabolism , Amino Acid Sequence , Aspergillus niger/genetics , Binding Sites , Biocatalysis , Catalytic Domain , Flavin-Adenine Dinucleotide/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Isomerism , Molecular Sequence Data , Monoamine Oxidase/genetics , Mutant Proteins/chemistry , Mutation/genetics , Oxidation-Reduction , Proline/metabolism , Protein Structure, Secondary , Sequence Alignment , Static Electricity , Substrate SpecificityABSTRACT
A template-based mnemonic has been developed for the enzyme monoamine oxidase from Aspergillus niger and has been used to successfully identify the alkaloid (+/-)-crispine A as a target for chemo-enzymatic deracemisation yielding the biologically active (R)-enantiomer in 97% e.e.
