The natural products beauveriolide I and III: a new class of beta-amyloid-lowering compounds.

Attacking Alzheimer's by ACAT: The aggregation of beta-amyloid peptides, especially Abeta(42), into senile plaques is a hallmark of Alzheimer's disease (AD). We show that the fungal natural products beauveriolides I and III can potently decrease Abeta secretion from cells expressing human amyloid precursor protein; this offers a potential new scaffold for the development of compounds with proven bioavailability for the treatment of AD.

A chemical approach to immunoprotein engineering: chemoselective functionalization of thioester proteins in their native state.

Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain complexed with small-molecule inhibitors highlight active-site flexibility.

The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

Emerging chemical and biological approaches for the preparation of discovery libraries.

The term of combinatorial chemistry has come to embrace all types of small-molecule library strategies. In both academic and industrial settings, the development of many combinatorial techniques has been driven by a desire to generate diverse libraries of molecules. Recently, chemical approaches have been reported that range from alternative library design concepts to new synthetic procedures that allow for selective product formation in extremely short reaction times. Concurrently, biological techniques have made great strides by adapting naturally occurring strategies for library preparation and screening. With proper understanding of library design, combinatorial endeavors and molecular libraries will continue to impact on the development of the next generation of tools for modern medicine.

Identification of a potent botulinum neurotoxin a protease inhibitor using in situ lead identification chemistry.

[reaction: see text] Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. By using in situ lead identification chemistry, we have uncovered the first class of inhibitors that displays nanomolar potency. From a 15 microM lead compound, structure-activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays an inhibition constant of 300 nM.

Identification of a botulinum neurotoxin A protease inhibitor displaying efficacy in a cellular model.

Herein we describe a small-molecule, non-peptidic, inhibitor for botulinum neurotoxin A protease, that displays for the first time efficacy in a cell-based assay.

A furanosyl-carbonate autoinducer in cell-to-cell communication of V. harveyi.

An autoinducer arising from reaction of cyclized S-DPD and carbonate is shown to induce light in V. harveyi and thus may play a previously unknown role in quorum sensing.

Cholesterol secosterol aldehydes induce amyloidogenesis and dysfunction of wild-type tumor protein p53.

Epidemiologic and clinical evidence points to an increased risk for cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and -B (ALD), but not the polyunsaturated fatty acid (PUFA)-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by the analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction and cancer risk.

Alpha- and beta-stilbenosides as base-pair surrogates in DNA hairpins.

The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective preparation of both the alpha- and beta-stilbenosides have been developed. Both anomers are effective in stabilizing hairpin structures when used as capping groups at the open end of the hairpin base-pair domain. However, only the beta-anomer effectively stabilizes the hairpin structure when located in the interior of the base-pair domain opposite an abasic site. Similar results are obtained for hairpins possessing two stilbenosides, either adjacent to each other or with one intervening base-pair. Molecular dynamics simulations are employed to obtain averaged structures for these conjugates. The calculated structures for the capped hairpins formed with either anomer show effective pi-stacking with the adjacent base-pair. The calculated structures for the internal stilbenosides show that the alpha- and beta-anomers form extrahelical and intrahelical structures, respectively. The relative orientations of the two stilbenes in the bis-stilbenosides have been studied using a combination of exciton-coupled circular dichroism spectroscopy and molecular modeling.

Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors.

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.

Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation.

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate.

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.

Altered retinoid homeostasis catalyzed by a nicotine metabolite: implications in macular degeneration and normal development.

Retinoids (vitamin A) serve two distinct functions in higher animals: light absorption for vision and gene regulation for growth and development. Cigarette smoking is a contributing factor for diseases that affect vision such as age-related macular degeneration and increases the risk of birth defects; however, altered retinoid homeostasis has received little attention as a potential mechanism for smoking-associated toxicities. Herein, we demonstrate that nornicotine, a nicotine metabolite and component of cigarette smoke, catalyzes the Z-to-E alkene isomerization of unsaturated aldehydes and ketones, including retinals. Despite the recent explosion in the use of organic compounds as chemical catalysts, minimal effort has been devoted to biologically relevant organocatalysis. Our study demonstrates a system in which a lowest unoccupied molecular orbital-lowering intermediate similar to the endogenous protein rhodopsin effectively catalyzes isomerization under biologically relevant conditions. The product of retinal isomerization is all-E-retinal, which in the eye is a biosynthetic precursor to N-retinylidene-N-retinylethanolamine, a hallmark of age-related macular degeneration. Furthermore, 9-Z- and all-E-retinal isomers are biosynthetic precursors to 9-Z- and all-E-retinoic acids, ligands that mediate specific cellular responses by binding to transcriptional regulatory proteins critical in growth and development. Strict maintenance of retinal isomer composition is essential for proper transcriptional regulation. Nornicotine-catalyzed retinal isomerization implies an underlying molecular mechanism for age-related macular degeneration, the birth defects associated with smoking, and other smoking-associated abnormalities that stem from disruption of retinoid metabolism.

Enantioreversal in the sharpless asymmetric epoxidation reaction controlled by the molecular weight of a covalently appended achiral polymer.

Polymers such as poly(ethylene glycol) (PEG) have proven use in a variety of applications including organic synthesis. We now disclose our investigations into the recently disputed report that PEG tartrate esters can reverse the enantioselectivity of the Sharpless asymmetric epoxidation reaction. The results presented herein have clarified that the enantioselectivity of this reaction can be reproducibly reversed solely as a function of the molecular weight of the appended PEG. By preparing a range of tartrate ligands with varying PEG chains lengths, the reversal was found to occur within a molecular weight change of only 800. As the PEG chain did not affect the inherent chirality of the ligand, the enantioreversal was proposed to occur as a result of two Ti-ligand complexes which differ in their molecularity of ligand, one monomeric in ligand and the other dimeric. Support for this hypothesis was given through equilibrium measurements which revealed that the predominant species in Ti/PEG tartrate ester mixtures is a distinct 2:1 Ti-ligand complex, as opposed to the 2:2 Ti-ligand complex of traditional Sharpless asymmetric epoxidations. In total, these data represent an unrecognized property of PEG-supported catalysts that could open up new venues in the control of asymmetric reactions by means of achiral appended polymers.