Leonard Favreau

The Rat Quinone Reductase Antioxidant Response Element IDENTIFICATION OF THE NUCLEOTIDE SEQUENCE REQUIRED FOR BASAL AND INDUCIBLE ACTIVITY AND DETECTION OF ANTIOXIDANT RESPONSE ELEMENT-BINDING PROTEINS IN HEPATOMA AND NON-HEPATOMA CELL LINES

The antioxidant response element (ARE) found in the 5′-flanking region of the rat quinone reductase gene has been further characterized by mutational and deletion analysis. The results indicate that the 31-base pair ARE, which contains a 13-base pair palindromic sequence, can be further separated into three regions, all three of which are required for elevated basal level gene expression. These three regions include the proximal and distal half-sites as well as a 3′-flanking region consisting of 4 adenine nucleotides. Neither the proximal nor the distal half-site alone mediates transcriptional activation by β-naphthoflavone. However, when placed together the two half-sites restore responsiveness to the inducer. Interestingly, the presence of only 1 of the 4 adenine nucleotides in the 3′-flanking region of the proximal half-site is required for responsiveness to the inducer. Point mutations within the ARE indicate that several nucleotides in both the proximal and distal half-sites are required for basal level gene expression. Electrophoretic mobility shift analysis using the ARE as the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from human Hep G2 cells. Further, this complex can be detected in nuclear extracts from rat liver and rat hepatoma cells but not in mouse Hepa 1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can also be detected in F9 cells which lack significant levels of Jun/Fos proteins. Although the rat ARE resembles the human quinone reductase ARE which contains a consensus TRE, the 2-nucleotide change in the core sequence (TGACTCA versus TGACTTG) eliminates the high affinity TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in Hep G2 and other cells with different properties than AP-1.

Discovery of Cyclic Acylguanidines as Highly Potent and Selective beta-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I-Inhibitor Design and Validation

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

Structure based design of iminohydantoin BACE1 inhibitors: Identification of an orally available, centrally active BACE1 inhibitor

From an initial lead 1, a structure-based design approach led to identification of a novel, high-affinity iminohydantoin BACE1 inhibitor that lowers CNS-derived Aβ following oral administration to rats. Herein we report SAR development in the S3 and F subsites of BACE1 for this series, the synthetic approaches employed in this effort, and in vivo data for the optimized compound.

Design and validation of bicyclic iminopyrimidinones as beta amyloid cleaving enzyme-1 (BACE1) inhibitors: conformational constraint to favor a bioactive conformation.

On the basis of our observation that the biaryl substituent of iminopyrimidinone 7 must be in a pseudoaxial conformation to occupy the contiguous S1-S3 subsites of BACE1, we have designed a novel fused bicyclic iminopyrimidinone scaffold intended to favor this bioactive conformation. Strategic incorporation of a nitrogen atom in the new constrained ring allowed us to develop SAR around the S2 binding pocket and ultimately resulted in analogues with low nanomolar potency for BACE1. In particular, optimization of the prime side substituent led to major improvements in potency by displacement of two conserved water molecules from a region near S2. Further optimization of the pharmacokinetic properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aβ(40) in the plasma, CSF, and cortex of rats in a dose-dependent manner.

Piperazine sulfonamide BACE1 inhibitors: design, synthesis, and in vivo characterization.

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2 sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Abeta(40) in transgenic mice with a single subcutaneous dose.

The Antioxidant Response Element

Oxidative stress results from the production of reactive oxygen intermediates, which can arise from a number of sources including ionizing radiation, inflammation, and electrophilic xenobiotics. Quinones are highly electrophilic compounds and can be very reactive, depending on the degree of reduction. Quinones can undergo either a one-electron reduction by NADPH-cytochrome P-450 reductase to form the semiquinone free radical, a reactive oxygen intermediate, or twoelectron reduction by NAD(P)H:quinone oxidoreductase (quinone reductase, DT-diaphorase) to form the hydroquinone, a less reactive species.1 In the presence of oxygen, the semiquinone autoxidizes to form the original quinone and Superoxide anion radical. The repetition of this reaction (oxidation-reduction) can lead to oxidative stress,2 which can cause cellular damage. One cellular defense mechanism against the toxic and neoplastic effects of quinones is believed to be the enzyme quinone reductase. Rat liver cytosol from 3-methylcholanthrene (3-MC) treated rats containing high levels of quinone reductase was shown to reduce the amount of semiquinone produced in vitro using menadione as the quinone acceptor. In contrast, addition of microsomes from phenobarbital-treated rats (which contain high levels of NADPH-cytochrome P-450 reductase) or cytosol containing dicoumerol, a potent quinone reductase inhibitor, resulted in production of the semiquinone.3 Thus quinone reductase, which produces hydroquinones that can be further metabolized by conjugation and then rapidly eliminated, can function as a cellular protective mechanism against oxidative stress from xenobiotics as well as endogenous compounds.4

Structure-Based Design of an Iminoheterocyclic β-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aβ in Nonhuman Primates

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2-S2″ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aβ40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aβ in nonrodent preclinical species.

Discovery of potent iminoheterocycle BACE1 inhibitors.

The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aβ40 levels upon subcutaneous and oral administration to rats.

The synthesis and structure–activity relationship of 4-benzimidazolyl-piperidinylcarbonyl-piperidine analogs as histamine H3 antagonists

A structure-activity relationship study of the lead piperazinylcarbonylpiperidine compound 3 resulted in the identification of 4-benzimidazolyl-piperidinylcarbonyl-piperidine 6h as a histamine-3 (H(3)) receptor antagonist. Additional optimization of 6h led to the identification of compounds 11i-k with K(i) <or= 0.5 nM and good in vivo activity.