Celastrol Analogues as Inducers of the Heat Shock Response. Design and Synthesis of Affinity Probes for the Identification of Protein Targets

The natural product celastrol (<b>1</b>) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by <i>in vitro</i> analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogues. Conversion of the carboxylic acid functionality to amides and long-chain analogues, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (<b>23</b> and <b>24</b>) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol

Mechanistic Studies of Inactivation of Inducible Nitric Oxide Synthase by Amidines

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and nitric oxide. <i>N</i>⁵-(1-Iminoethyl)-l-ornithine (l-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate l-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of l-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the l-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on <b>10</b> and <b>11</b> to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (l-NIO), the other alkyl groups block binding of O₂ at the heme iron. Compounds <b>8</b>, <b>9</b>, and <b>11</b> were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl <b>8</b>. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The l-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of l-NIO, and the inactivation mechanism previously reported for l-NIO appears to be unique to methyl amidines

Design and Synthesis of Potent Quinazolines as Selective β‑Glucocerebrosidase Modulators

Gaucher’s disease is a common genetic disease caused by mutations in the β-glucocerebrosidase (GBA1) gene that have been also linked to increased risk of Parkinson’s disease and Lewy body dementia. Stabilization of misfolded mutant β-glucocerebrosidase (GCase) represents an important therapeutic strategy in synucleinopathies. Here we report a novel class of GCase quinazoline inhibitors, obtained in a high throughput screening, with moderate potency against wild-type GCase. Rational design and a SAR study of this class of compounds led to a new series of quinazoline derivatives with single-digit nanomolar potency. These compounds were shown to selectively stabilize GCase when compared to other lysosomal enzymes and to increase N370S mutant GCase protein concentration and activity in cell assays. To the best of our knowledge, these molecules are the most potent noniminosugar GCase modulators to date that may prove useful for future mechanistic studies and therapeutic approaches in Gaucher’s and Parkinson’s diseases

Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2‑Aminoquinoline Inhibitors

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, the Caco-2 assay also revealed improved membrane permeability over previous compounds

Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2‑Aminoquinoline Inhibitors

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, the Caco-2 assay also revealed improved membrane permeability over previous compounds

Total Synthesis of Tambromycin Enabled by Indole C–H Functionalization

The total synthesis of tambromycin (<b>1</b>), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (<b>11</b>). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C–H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline

Methylated <i>N</i>^ω‑Hydroxy‑l‑arginine Analogues as Mechanistic Probes for the Second Step of the Nitric Oxide Synthase-Catalyzed Reaction

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline through the intermediate <i>N</i>^ω-hydroxy-l-arginine (NHA), producing nitric oxide, an important mammalian signaling molecule. Several disease states are associated with improper regulation of nitric oxide production, making NOS a therapeutic target. The first step of the NOS reaction has been well-characterized and is presumed to proceed through a compound I heme species, analogous to the cytochrome P450 mechanism. The second step, however, is enzymatically unprecedented and is thought to occur via a ferric peroxo heme species. To gain insight into the details of this unique second step, we report here the synthesis of NHA analogues bearing guanidinium methyl or ethyl substitutions and their investigation as either inhibitors of or alternate substrates for NOS. Radiolabeling studies reveal that <i>N</i>^ω-methoxy-l-arginine, an alternative NOS substrate, produces citrulline, nitric oxide, and methanol. On the basis of these results, we propose a mechanism for the second step of NOS catalysis in which a methylated nitric oxide species is released and is further metabolized by NOS. Crystal structures of our NHA analogues bound to nNOS have been determined, revealing the presence of an active site water molecule only in the presence of singly methylated analogues. Bulkier analogues displace this active site water molecule; a different mechanism is proposed in the absence of the water molecule. Our results provide new insights into the steric and stereochemical tolerance of the NOS active site and substrate capabilities of NOS

Improvement of Cell Permeability of Human Neuronal Nitric Oxide Synthase Inhibitors Using Potent and Selective 2‑Aminopyridine-Based Scaffolds with a Fluorobenzene Linker

Inhibition of neuronal nitric oxide synthase (nNOS) is a promising therapeutic approach to treat neurodegenerative diseases. Recently, we have achieved considerable progress in improving the potency and isoform selectivity of human nNOS inhibitors bearing a 2-aminopyridine scaffold. However, these inhibitors still suffered from too low cell membrane permeability to enter into CNS drug development. We report herein our studies to improve permeability of nNOS inhibitors as measured by both PAMPA–BBB and Caco-2 assays. The most permeable compound (<b>12</b>) in this study still preserves excellent potency with human nNOS (<i>K</i>_i = 30 nM) and very high selectivity over other NOS isoforms, especially human eNOS (hnNOS/heNOS = 2799, the highest hnNOS/heNOS ratio we have obtained to date). X-ray crystallographic analysis reveals that <b>12</b> adopts a similar binding mode in both rat and human nNOS, in which the 2-aminopyridine and the fluorobenzene linker form crucial hydrogen bonds with glutamate and tyrosine residues, respectively

Design and Evaluation of 3‑(Benzylthio)benzamide Derivatives as Potent and Selective SIRT2 Inhibitors

Inhibitors of sirtuin-2 (SIRT2) deacetylase have been shown to be protective in various models of Huntington’s disease (HD) by decreasing polyglutamine aggregation, a hallmark of HD pathology. The present study was directed at optimizing the potency of SIRT2 inhibitors containing the 3-(benzylsulfonamido)­benzamide scaffold and improving their metabolic stability. Molecular modeling and docking studies revealed an unfavorable role of the sulfonamide moiety for SIRT2 binding. This prompted us to replace the sulfonamide with thioether, sulfoxide, or sulfone groups. The thioether analogues were the most potent SIRT2 inhibitors with a two- to three-fold increase in potency relative to their corresponding sulfonamide analogues. The newly synthesized compounds also demonstrated higher SIRT2 selectivity over SIRT1 and SIRT3. Two thioether-derived compounds (<b>17</b> and <b>18</b>) increased α-tubulin acetylation in a dose-dependent manner in at least one neuronal cell line, and <b>18</b> was found to inhibit polyglutamine aggregation in PC12 cells

Accessible Chiral Linker to Enhance Potency and Selectivity of Neuronal Nitric Oxide Synthase Inhibitors

The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily synthesized compounds containing either one (<b>20a</b>,<b>b</b>) or two (<b>9a</b>–<b>d</b>; <b>15a</b>,<b>b</b>) 2-amino-4-methylpyridine groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor <b>9c</b> is the best of these compounds, having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS complexed with either <b>9a</b> or <b>9c</b> show a double-headed binding mode, where each 2-aminopyridine headgroup interacts with either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of <b>9c</b> contributes additional hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from the pyrrolidine of <b>9a</b> to the heme propionate allows the inhibitor to adopt two different binding orientations. Both <b>9a</b> and <b>9c</b> bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity