Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis.

The Erm family of methyltransferases confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics through the methylation of 23S ribosomal RNA. Upon the methylation of RNA, the MLS antibiotics lose their ability to bind to the ribosome and exhibit their antibiotic activity. Using an NMR-based screen, we identified a series of triazine-containing compounds that bind weakly to ErmAM. These initial lead compounds were optimized by the parallel synthesis of a large number of analogues, resulting in compounds which inhibit the Erm-mediated methylation of rRNA in the low micromolar range. NMR and X-ray structures of enzyme/inhibitor complexes reveal that the inhibitors bind to the S-adenosylmethionine binding site on the Erm protein. These compounds represent novel methyltransferase inhibitors that serve as new leads for the reversal of Erm-mediated MLS antibiotic resistance.

Potent and orally bioavailable noncysteine-containing inhibitors of protein farnesyltransferase.

Potent and orally bioavailable nonthiol-containing inhibitors of protein farnesyltransferase are described. Oral bioavailability was achieved by replacement of the pyridyl ether moiety of 1 with a 2-substituted furan ether to give 4. Potency was regained with 2,5-disubstituted furan ethers while maintaining the bioavailability inherent in 4. p-Chlorophenylfuran ether 24 is 0.7 nM in vitro (FTase) and is 32% bioavailable in the mouse, 30% bioavailable in rats, and 21% bioavailable in dogs.

NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio.

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. Here we describe the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two a helices. In addition, we have discovered a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins.

Potent and selective non-cysteine-containing inhibitors of protein farnesyltransferase.

Potent and selective non-thiol-containing inhibitors of protein farnesyltransferase are described. FTI-276 (1) was transformed into pyridyl ether analogue 19. The potency of pyridyl ether 19 was improved by modification of the biphenyl core to that of an o-tolyl substituted biphenyl core to give 29. In addition to 0.4 nM in vitro potency, 29 displayed 350 nM potency in whole cells as the parent carboxylic acid. The o-tolyl biphenyl core dramatically and unexpectedly enhanced the potency of other compounds as exemplified by 46, 47, 48, and 49.

Design, synthesis, and in vitro evaluation of cyclic nitrones as free radical traps for the treatment of stroke.

Analogs of the cyclic nitrone free radical trap 1 (3,3-dimethyl-3,4-dihydroisoquinoline N-oxide, a cyclic analog of phenyl-tert-butylnitrone (PBN)) were prepared in which (1) the fused phenyl ring was replaced with a naphthalene ring, an electron rich heterocycle, or a dimethylphenol, (2) the nitrone-containing ring comprised five, six, or seven atoms, and (3) the gem-dimethyl group was replaced with spirocyclic groups. The most active antioxidant, which bears a dimethylphenol fused to a 7-membered ring nitrone (compound 6h), inhibited lipid peroxidation in vitro with an IC50 of 22 microM, a 75-fold improvement over that of 1. The previously observed correlation between lipophilicity and activity vs lipid peroxidation in vitro has been further substantiated and refined by this study. Moreover, certain classes of compounds (namely, dimethylphenols 6g,h and furan 6j) have now been found which are considerably more active in vitro than expected on the basis of their log k'(w) values.

Cyclic nitrone free radical traps: isolation, identification, and synthesis of 3,3-dimethyl-3,4-dihydroisoquinolin-4-ol N-oxide, a metabolite with reduced side effects.

A C-4 hydroxylated metabolite (2, 3,3-dimethyl-3,4-dihydroisoquinolin-4-ol N-oxide) of the previously described cyclic nitrone free radical trap 1 (3,3-dimethyl-3,4-dihydroisoquinoline N-oxide, a cyclic analog of phenyl-tert-butylnitrone (PBN)) was isolated, identified, and synthesized. The metabolite (2), though a less potent antioxidant than 1 in an in vitro lipid peroxidation assay, showed greatly reduced acute toxicity and sedative properties. Several analogs of 2 were prepared in attempts to improve on its weak antioxidant activity while retaining the desirable side effect profile. Effective structural changes included replacement of the gem-dimethyl moiety with spirocycloalkane groups and/or oxidation of the alcohols to the corresponding ketones. All of the analogs were more lipophilic (log k'(w)) and more active in the standard lipid peroxidation assay than 2. In addition, some of the compounds were able to protect cerebellar granule cells against oxidative damage (an in vitro model of oxidative brain injury) with IC50 values well below the value of the lead compound 1. The ketones, as predicted, were much more potent than 2 (and 1) in both of the above assays (up to ca. 200-fold). However, only compounds with a hydroxyl or an acetate group at C-4 displayed significantly reduced acute toxicity and sedative properties relative to those of 1. Importantly, the diminishment of toxicity and sedation were not the result of a lack of brain penetration as both 2 and the corresponding ketone (3,3-dimethyl-3,4-dihydro-3H-isoquinolin-4-one N-oxide) achieved equal or greater brain levels than those of 1 when administered to rats i.p.