Richard B. Silverman

Celastrols as inducers of the heat shock response and cytoprotection

Alterations in protein folding and the regulation of conformational states have become increasingly important to the functionality of key molecules in signaling, cell growth, and cell death. Molecular chaperones, because of their properties in protein quality control, afford conformational flexibility to proteins and serve to integrate stress-signaling events that influence aging and a range of diseases including cancer, cystic fibrosis, amyloidoses, and neurodegenerative diseases. We describe here characteristics of celastrol, a quinone methide triterpene and an active component from Chinese herbal medicine identified in a screen of bioactive small molecules that activates the human heat shock response. From a structure/function examination, the celastrol structure is remarkably specific and activates heat shock transcription factor 1 (HSF1) with kinetics similar to those of heat stress, as determined by the induction of HSF1 DNA binding, hyperphosphorylation of HSF1, and expression of chaperone genes. Celastrol can activate heat shock gene transcription synergistically with other stresses and exhibits cytoprotection against subsequent exposures to other forms of lethal cell stress. These results suggest that celastrols exhibit promise as a new class of pharmacologically active regulators of the heat shock response.

Mechanism-based enzyme inactivators.

Publisher Summary An enzyme inactivator, in general, is a compound that produces irreversible inhibition of the enzyme— that is, it irreversibly prevents the enzyme from catalyzing its reaction. Irreversible in this context, however, does not necessarily mean that the enzyme activity never returns only that the enzyme becomes dysfunctional for an extended (but unspecified) period of time. A mechanism-based enzyme inactivator, by the definition used here, is a compound that is transformed by the catalytic machinery of the enzyme into a species that acts as an affinity labeling agent, a transition state analog, or a tight-binding inhibitor (either covalent or noncovalent) prior to release from the enzyme. Mechanism-based enzyme inactivation is a powerful tool for the studies of enzyme mechanisms and mechanisms of enzyme inactivation, by small molecules. Mechanistic hypotheses can be tested by appropriate molecular design, utilizing the isotopically labeled analogs, to permit the elucidation of the structures of metabolites produced and to determine the portions of the mechanism-based inactivators that become covalently attached to the target enzyme. This approach to the studies of enzyme mechanisms is well suited for those who are geared more to the organic chemistry of enzymecatalyzed reactions and who have insights into the chemical machinery of active sites of enzymes. The use of mechanism-based enzyme inactivators is yet another of the very important methods in enzymology.

Activation of Heat Shock and Antioxidant Responses by the Natural Product Celastrol: Transcriptional Signatures of a Thiol-targeted Molecule

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrols biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.

The Sirtuin 2 Inhibitor AK-7 Is Neuroprotective in Huntington’s Disease Mouse Models

Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinsons disease and Huntingtons disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans.

3-alkyl GABA and 3-alkylglutamic acid analogues: two new classes of anticonvulsant agents.

Recently we showed that 3-alkyl-4-aminobutanoic acids are in vitro activators of brain L-glutamic acid decarboxylase (GAD) that show anticonvulsant activity. Since activation of GAD leads to increased concentrations of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in vitro, these compounds could represent a new class of anticonvulsant agents. Here it is shown that 3-alkylglutamic acid analogues also activate GAD and that all of the compounds in both series are active anticonvulsant agents against low intensity electroshock in mice. The most active compound, 3-isobutyl GABA, was tested further against maximal electroshock in mice and was shown to be very potent after both intravenous and oral administration without causing ataxia. It is not known if brain GABA levels are elevated in vivo by administration of these compounds or if the mechanism of anticonvulsant activity is related to their ability to activate GAD.

Revisiting heme mechanisms. A perspective on the mechanisms of nitric oxide synthase (NOS), Heme oxygenase (HO), and cytochrome P450s (CYP450s).

Despite the essential biological importance of reactions that involve heme, mechanisms of heme reactions in enzymes like nitric oxide synthase (NOS), heme oxygenase (HO), and cytochrome P450s (CYP450s) are still not well-understood. This Perspective on NOS, HO, and CYP450 mechanisms is written from the point of view of the heme chemistry. Steps in the classical heme catalytic cycle are discussed based on the specific environment within each of these enzymes. Elucidation of the mechanisms of NOS inactivation by some substrate analogues provides important mechanistic clues to the NOS catalytic mechanism. On the basis of mechanistic studies of NOS inactivation by amidine analogues of l-arginine and other previous mechanistic results, a new mechanism for NOS-catalyzed l-arginine NG-hydroxylation (the first half of the catalytic reaction) is proposed in this Perspective. The key step in the second half of the NOS catalytic reaction, the internal electron transfer between the substrate and heme, is discussed on the basis of mechanistic results of NOS inactivation by NG-allyl-l-arginine and the structures of the substrate intermediates. Elucidation of the mechanism of NOS inactivation by amidines, which leads to heme degradation, also provides important mechanistic implications for heme oxygenase-catalyzed heme catabolism. Focusing on the meso-hydroxylation step during inactivation of NOS by amidines as well as the HO-catalyzed reaction, the essential nature of the heme-oxygen species responsible for porphyrin meso-hydroxylation is discussed. Finally, on the basis of the proposed heme degradation mechanism during NOS inactivation and the HO-catalyzed reaction, the mechanism for the formation of the monooxygenated heme species in P450-catalyzed reactions is discussed.

Structures of GABA aminotransferase, a pyridoxal 5'-phosphate and [2Fe-2S] cluster containing enzyme, complexed with -EthynylGABA and with the antiepilepsy drug vigabatrin

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, γ-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue γ-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5′-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

From Basic Science to Blockbuster Drug: The Discovery of Lyrica

Many great discoveries occur when they are least expected. These discoveries are most satisfying when they derive from studies of basic science. That is how the new blockbuster drug for the treatment of various neuropathic pains, epilepsy, and generalized anxiety disorder, Lyrica (pregabalin), was discovered. This essay describes the discovery and features of this new drug. One of the early projects I initiated when I started my independent career at Northwestern University was the design and mechanism of new inactivators of the pyridoxal 5’-phosphate (PLP)-dependent enzyme g-aminobutyric acid aminotransferase (GABA-AT). GABA-AT is the enzyme responsible for the degradation of the inhibitory neurotransmitter, GABA, leading to its conversion to the excitatory neurotransmitter l-glutamate. Compounds that inhibit this enzyme have anticonvulsant activity as well as exhibit activity against Huntington0s disease, Alzheimer0s disease, Parkinson0s disease, and drug addiction. Epilepsy, broadly defined as any disease characterized by recurring convulsive seizures, has been known for many millennia. There are numerous etiologies for epilepsy because it is not a single disease; consequently, 1–2% of the world population has some form of epilepsy. Of those afflicted with this disease, 30–40% do not respond to multiple anticonvulsant drugs. There are many causes for seizures, but one of them is an imbalance in the concentration of GABA relative to l-glutamate. When GABA levels in the brain diminish, seizures can result. Injection of GABA directly into the brain can terminate the seizure, but administration of GABA, either orally or intravenously, has no effect because GABA, a hydrophilic charged molecule, does not cross the blood–brain barrier (BBB), a membrane that protects the brain from chemicals in the blood while still allowing essential metabolic function. The BBB comprises very tightly packed endothelial cells, which provide the walls of the blood vessels perfusing the brain; this higher density of cells restricts passage of unwanted substances from the bloodstream into the brain. One approach to increase the GABA concentration in the brain is to design a compound that can cross the BBB and inhibit GABA-AT, the only enzyme that degrades brain GABA. This prevents the breakdown of GABA, and its concentration rises, resulting in an anticonvulsant effect. Because of the importance of increasing brain GABA levels in central nervous system (CNS) disorders, my group, in the years 1981–1988, designed a series of mechanism-based inactivators for GABA-AT. It became apparent, however, that to progress toward the design of a new anticonvulsant agent, it would be necessary to prepare compounds that were selective inhibitors of GABA-AT (to raise GABA levels) without inhibiting l-glutamic acid decarboxylase (GAD), the PLP-dependent enzyme that converts the excitatory neurotransmitter, l-glutamate, to the inhibitory neurotransmitter, GABA (Scheme 1). Inhibition of GAD would decrease the concentration of GABA, the opposite of the desired effect. Furthermore, for brain penetration, which would be required for an anticonvulsant drug, increased lipophilicity would be important. Consequently, in 1988 I asked Dr. Ryszard Andruszkiewicz, a visiting scholar from the Technical University of Gdansk, to synthesize a series of 3alkyl-GABA and 3-alkylglutamate analogues, then to measure their inhibition of GABA-ATand GAD to determine if we could identify more lipophilic analogues that selectively bound to the former and not the latter enzyme. Dr. Andruskiewicz proceeded to synthesize fourteen 3-alkyl-GABA analogues (including four stereoisomers) (Scheme 2), 4-methyl-GABA (and its two enantiomers) and seven 3-alkylglutamate analogues. All of the GABA analogues were substrates for GABAAT. As the substituent size increased, so did the Michaelis constant Km; the Vmax/Km value for the 3-methyl analogue was a little larger than that for GABA, but the Vmax/Km values for the remaining analogues were progressively smaller (Vmax is the maximum velocity of the enzymatic reaction). The unexpected surprise came when these compounds were tested as inhibitors for GAD. Not only was none of them an inhibitor, but all of them were found to activateGAD, that is, the addition of compound produced an increased rate of GABA formation (Figure 1)! This had not previously been observed. My immediate thought was to have these compounds tested as anticonvulsant agents because they might provide a new [*] R. B. Silverman John Evans Professor of Chemistry Department of Chemistry Department of Biochemistry, Molecular Biology, and Cell Biology Center for Drug Discovery and Chemical Biology, Northwestern University 2145 Sheridan Road, Evanston, Illinois 60208-3113 (USA) Fax: (+1)847-491-7713 E-mail: Agman@chem.northwestern.edu Essays

Target- and mechanism-based therapeutics for neurodegenerative diseases: Strength in numbers

The development of new therapeutics for the treatment of neurodegenerative pathophysiologies currently stands at a crossroads. This presents an opportunity to transition future drug discovery efforts to target disease modification, an area in which much still remains unknown. In this Perspective we examine recent progress in the areas of neurodegenerative drug discovery, focusing on some of the most common targets and mechanisms: N-methyl-d-aspartic acid (NMDA) receptors, voltage gated calcium channels (VGCCs), neuronal nitric oxide synthase (nNOS), oxidative stress from reactive oxygen species, and protein aggregation. These represent the key players identified in neurodegeneration and are part of a complex, intertwined signaling cascade. The synergistic delivery of two or more compounds directed against these targets, along with the design of small molecules with multiple modes of action, should be explored in pursuit of more effective clinical treatments for neurodegenerative diseases.

Reduced thioredoxin: A possible physiological cofactor for vitamin k epoxide reductase. further support for an active site disulfide

Vitamin K 2,3-epoxide reductase activity from liver microsomes requires only a thiol cofactor, particularly dithiothreitol (DTT). In order to identify a likely physiological cofactor, reduced lipoic acid and reduced thioredoxin were tested as cofactors in beef and rat liver microsomal systems. Reduced lipoic acid is only about one-third as active as DTT in both systems. Thioredoxin, however, is significantly more active than either DTT or reduced lipoic acid in both systems; thioredoxin binds 188 times better than does DTT. The thioredoxin must be in the reduced form since omission of either thioredoxin reductase or NADPH results in complete loss of enzyme activity. The concentration of DTT required to obtain maximal enzyme activity may be as much as 485 times greater than the corresponding concentration of reduced thioredoxin that gives the same enzyme activity.