Alzheimer's disease is the leading cause of dementia among the elderly, and with the ever-increasing size of this population, cases of Alzheimer's disease are expected to triple over the next 50 years. Consequently, the development of treatments that slow or halt the disease progression have become imperative to both improve the quality of life for patients and reduce the health care costs attributable to Alzheimer's disease. Here, we demonstrate that the active component of marijuana, Delta9-tetrahydrocannabinol (THC), competitively inhibits the enzyme acetylcholinesterase (AChE) as well as prevents AChE-induced amyloid beta-peptide (Abeta) aggregation, the key pathological marker of Alzheimer's disease. Computational modeling of the THC-AChE interaction revealed that THC binds in the peripheral anionic site of AChE, the critical region involved in amyloidgenesis. Compared to currently approved drugs prescribed for the treatment of Alzheimer's disease, THC is a considerably superior inhibitor of Abeta aggregation, and this study provides a previously unrecognized molecular mechanism through which cannabinoid molecules may directly impact the progression of this debilitating disease.

The forte of catalytic antibodies has resided in the control of the ground-state reaction coordinate. A principle and method are now described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces. The key component is a chemically reactive optical sensor that provides a direct report of the dynamic interplay between protein and ligand at the active site. To illustrate the concept, we used a trans-stilbene hapten to elicit a panel of monoclonal antibodies that displayed a range of fluorescent spectral behavior when bound to a trans-stilbene substrate. Several antibodies yielded a blue fluorescence indicative of an excited-state complex or "exciplex" between trans-stilbene and the antibody. The antibodies controlled the isomerization coordinate of trans-stilbene and dynamically coupled this manifold with an active-site residue. A step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.

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The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-Å resolution. The antibodybound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining͞distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.M odern theories of biological catalysis date from Haldane's theory of strain: ''using Fischer's lock and key simile, the key does not fit the lock perfectly but exercises a certain strain on it'' (1). Although the notion that enzymes use binding energy to strain or distort substrates is a fundamental theory of enzyme catalysis (2), it has proven difficult to experimentally validate (3). One approach that has proven effective in testing theories of biological catalysis involves the use of the programmable nature of antibody binding energy to evolve selective catalysts. Antibody catalysis has allowed us to dissect the energetic contributions of transition state stabilization, general base and covalent catalysis, and proximity effects to catalysis (4-9, 29). More recently, we showed that an antibody (7G12) raised against a strained ground-state mimic acted as an efficient porphyrin metallation catalyst (10). We now report the x-ray crystal structure of the Michaelis complex formed between the Fab fragment of the ferrochelatase antibody 7G12 and its substrate mesoporphyrin IX (MP), in which the bound substrate is distorted toward the transition-state conformation for metal insertion. The structure of this complex provides unequivocal structural evidence for the strain theory proposed by Haldane more than 70 years ago. Moreover, the detailed structural and biophysical characterization of the germ-line and affinity-matured antibodies has provided a detailed mechanistic picture of the immunological evolution of this strain mechanism.The enzyme ferrochelatase catalyzes the insertion of Fe 2ϩ into protoporphyrin IX as the last step in heme biosynthesis pathway (11). It was proposed that the enzyme catalyzes the porphyrin metallation reaction by distorting the porphyrin substrate toward a transition state-like geometry in which the pyrrole nitrogen lone pairs are exposed for metal chelation (12). To test this notion, antibody 7G12 was generated against N-methylmesoporphyrin (NMP) in which the porphyrin macrocycle is distorted due to alkylation at one of the pyrrole nitrogens. The resulting antibody was found to catalyze the metallation of MP by Zn 2ϩ with a catalytic efficiency comparable to that of the natural enzyme (10). The same antibody also catal...

Protein phosphatase 2C (PP2C) is an archetype of the PPM Ser/Thr phosphatases, characterized by dependence on divalent magnesium or manganese cofactors, absence of known regulatory proteins, and resistance to all known Ser/Thr phosphatase inhibitors. We have used virtual ligand screening with the AutoDock method and the National Cancer Institute Diversity Set to identify small molecule inhibitors of PP2Cα activity at a protein substrate. These inhibitors are active in the micromolar range, and represent the first non-phosphate-based molecules found to inhibit a type 2C phosphatase. The compounds docked to three recurrent binding sites near the PP2Cα active site and displayed novel Ser/Thr phosphatase selectivity profiles. Common chemical features of these compounds may form the basis for development of a PP2C inhibitor pharmacophore and may facilitate investigation of PP2C control and cellular function.

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

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The formation of beta-amyloid plaques in the brain is a key neurodegenerative event in Alzheimer's disease. Small molecules capable of binding to the peripheral anionic site of acetylcholinesterase (AChE) have been shown to inhibit the AChE-induced aggregation of the beta-amyloid peptide. Using the combination of a computational docking model and experimental screening, five compounds that completely blocked the amyloidogenic effect of AChE were rapidly identified from an approximately 200-member library of compounds designed to disrupt protein-protein interactions. Critical to this docking model was the inclusion of two explicit water molecules that are tightly bound to the enzyme. Interestingly, none of the tested compounds inhibited the related enzyme butyrylcholinesterase (BuChE) up to their aqueous solubility limits. These compounds are among the most potent inhibitors of amyloid beta-peptide aggregation and are equivalent only to propidium, a well-characterized AChE peripheral anionic site binder and aggregation inhibitor.