RESUMO
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the S-adenosyl-l-methionine (SAM)-dependent methylation of norepinephrine to form epinephrine. Epinephrine is implicated in the regulation of blood pressure, respiration, Alzheimer's disease, and post-traumatic stress disorder (PTSD). Transition-state (TS) analogues bind their target enzymes orders of magnitude more tightly than their substrates. A synthetic strategy for first-generation TS analogues of human PNMT (hPNMT) permitted structural analysis of hPNMT and revealed potential for second-generation inhibitors [Mahmoodi, N.; J. Am. Chem. Soc. 2020, 142, 14222-14233]. A second-generation TS analogue inhibitor of PNMT was designed, synthesized, and characterized to yield a Ki value of 1.2 nM. PNMT isothermal titration calorimetry (ITC) measurements of inhibitor 4 indicated a negative cooperative binding mechanism driven by large favorable entropic contributions and smaller enthalpic contributions. Cell-based assays with HEK293T cells expressing PNMT revealed a cell permeable, intracellular PNMT inhibitor with an IC50 value of 81 nM. Structural analysis demonstrated inhibitor 4 filling catalytic site regions to recapitulate both norepinephrine and SAM interactions. Conformation of the second-generation inhibitor in the catalytic site of PNMT improves contacts relative to those from the first-generation inhibitors. Inhibitor 4 demonstrates up to 51,000-fold specificity for PNMT relative to DNA and protein methyltransferases. Inhibitor 4 also exhibits a 12,000-fold specificity for PNMT over the α2-adrenoceptor.
Assuntos
Norepinefrina , Feniletanolamina N-Metiltransferase , Humanos , Feniletanolamina N-Metiltransferase/química , Feniletanolamina N-Metiltransferase/metabolismo , Células HEK293 , Epinefrina , Domínio CatalíticoRESUMO
The O-acetylation of the muramic acid residues in peptidoglycan (PG) is a modification that protects the bacteria from lysis due to the action of lysozyme. In Gram-negative bacteria, deacetylation is required to allow lytic transglycosylases to promote PG cleavage during cell growth and division. This deacetylation is catalyzed by O-acetylpeptidoglycan esterase (Ape) which is a serine esterase and employs covalent catalysis via a serine-linked acyl enzyme intermediate. Loss of Ape activity affects the size and shape of bacteria and dramatically reduces virulence. In this work, we report the first rationally designed aldehyde-based inhibitors of Ape from Campylobacter jejuni. The most potent of these acts as a competitive inhibitor with a Ki value of 13â µM. We suspect that the inhibitors are forming adducts with the active site serine that closely mimic the tetrahedral intermediate of the normal catalytic cycle. Support for this notion is found in the observation that reduction of the aldehyde to an alcohol effectively abolishes the inhibition.
Assuntos
Acetilesterase , Hominidae , Animais , Peptidoglicano/química , Aldeídos/farmacologia , Esterases/química , Bactérias/metabolismo , Serina , Hominidae/metabolismoRESUMO
Phenylethanolamine N-methyltransferase (PNMT) is a critical enzyme in catecholamine synthesis. It transfers the methyl group of S-adenosylmethionine (SAM) to catalyze the synthesis of epinephrine from norepinephrine. Epinephrine has been associated with diverse human processes, including the regulation of blood pressure and respiration, as well as neurodegeneration found in Alzheimer's disease. Human PNMT (hPNMT) proceeds through an SN2 transition state (TS) in which the transfer of the methyl group is rate limiting. TS analogue enzyme inhibitors are specific for their target and bind orders of magnitude more tightly than their substrates. Molecules resembling the TS of hPNMT were designed, synthesized, and kinetically characterized. This new inhibitory scaffold was designed to mimic the geometry and electronic properties of the hPNMT TS. Synthetic efforts resulted in a tight-binding inhibitor with a Ki value of 12.0 nM. This is among the first of the TS analogue inhibitors of methyltransferase enzymes to show an affinity in the nanomolar range. Isothermal titration calorimetry (ITC) measurements indicated negative cooperative binding of inhibitor to the dimeric protein, driven by favorable entropic contributions. Structural analysis revealed that inhibitor 3 binds to hPNMT by filling the catalytic binding pockets for the cofactor (SAM) and the substrate (norepinephrine) binding sites.
Assuntos
Inibidores Enzimáticos/farmacologia , Feniletanolamina N-Metiltransferase/antagonistas & inibidores , Calorimetria , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Feniletanolamina N-Metiltransferase/química , Feniletanolamina N-Metiltransferase/metabolismoRESUMO
An inhibitor bearing a phosphinylphosphonate group appended to a guanidinium functionality was designed to inhibit enzymes that generate carbocations from dimethylallyl diphosphate. When tested against human farnesyl diphosphate synthase the inhibitor bound with high micromolar affinity and did not bind more tightly than an isosteric inhibitor lacking the guanidinium functionality. When tested against the Type I isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Escherichia coli, the inhibitor bound with a Ki value of 120 nM, which was 400 times greater than its isosteric counterpart. This strategy of inhibition was much more effective with an enzyme that generates a carbocation that is not stabilized by both resonance and ion pairing, presumably because there is more evolutionary pressure on the enzyme to stabilize the cation.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Guanidina/farmacologia , Hemiterpenos/antagonistas & inibidores , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Geraniltranstransferase/antagonistas & inibidores , Geraniltranstransferase/metabolismo , Guanidina/síntese química , Guanidina/química , Hemiterpenos/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The indole prenyltransferase FtmPT1 catalyzes the C-2 normal prenylation of brevianamide F (cyclo-L-Trp-L-Pro) to give tryprostatin B. A previous structural analysis and studies with alternate substrates suggest that the reaction might not proceed through a direct C-2 attack, but could involve a C-3 prenylation followed by a rearrangement. In this work we investigated the reactivity of FtmPT1 with tryptophan, 5-hydroxybrevianamide, and 2-methylbrevianamide, and isolated products that had been reverse prenylated at C-3 and normal prenylated at N-1, C-3, or C-4. The formation of these products can be rationalized through mechanisms involving either an initial C-3 normal or C-3 reverse prenylation. In addition, we demonstrate that a C-3 reverse prenylated indole can undergo a nonenzymatic aza-Cope rearrangement at 37 °C to give an N-1 normal prenylated product. Together, these studies broaden the known product scope of this interesting catalyst and suggest that alternative mechanisms might be operating.
Assuntos
Alcaloides/química , Alcaloides/metabolismo , Biocatálise , Dimetilaliltranstransferase/metabolismo , Indóis/metabolismo , Aspergillus fumigatus/enzimologia , Domínio Catalítico , Dimetilaliltranstransferase/química , Modelos Moleculares , PrenilaçãoRESUMO
The structure of the title compound, C(26)H(28)N(2)O(2), contains essentially planar quinoline and benzene rings, the maximum deviations from the best plane being 0.086â (2) and 0.0056â (19)â Å, respectively; the dihedral angle between the rings is 82.87â (4)°. The adamantane cage consists of three fused cyclo-hexane rings in classical chair conformations, with C-C-C angles in the range 107.85â (15)-111.35â (15)°. Enanti-omers are linked alternately into chains along the c axis via short N-Hâ¯O inter-actions and further C-Hâ¯π inter-actions stabilize pairs of enanti-omers, forming a two-dimensional network.