Benzamides binding site

Analysis of the Benzamide Binding Site using Radioligand Binding Assays... 

Two non-competitive antagonists of 5-HT4-RS in guinea-pig ileum have also been described R 50595 [57, 58], a benzamide derivative (Table 2B) and FK 1052 a dihydropyridoindole derivative [59]. The mode of action of these drugs is not known. Do they pseudo-irreversibly bind to the agonist binding site, or to an allosteric site, or do they block the contraction by acting downstream to the 5-HT4-R ... 

The covalent binding property of benzamides has been exploited in the development of cell-based competitive binding assays that measure the ability of other antitubulin agents to inhibit binding of radiolabeled benzamides to j -tubulin in whole cells. The tritiated S-enantiomer of zoxamide has been used to study the zoxamide binding site in the Oomycete Phytophthora capsid [7]. Tritiated analogs 2 and 3 (RH-4032 and RH-5854, Fig. 16.1.2) have been used in similar assays in plant [4] and mammalian cells [6], respectively. 

The experimental benzamide fungicide zarilamide (4, Fig. 16.1.3 below) was discovered by ICI in the 1980s [8] and was found to act on microtubules [9]. Zarilamide was later shown to inhibit H-(S)-zoxamide binding to j -tubulin in Phytophthora capsid in a competitive manner [7], indicating a common binding site with zoxamide. 

In tobacco cells, the antimitotic herbicides pronamide (5) and the NPC chlor-propham (6) inhibited binding of H-RH-4032 to tobacco tubulin, suggesting a common binding site with zoxamide-related benzamides [4]. Similarly, colchicine... 

We have observed a stimulation of ADP-ribosylation by nanomolar concentrations of benzamides both in intact and permeabilized cells. This stimulation is reminiscent of that found with competitive inhibitors of allosteric enzymes. At very low NAD " concentrations, Kun has reported finding a sigmoidal curve of velocity versus NAD concentration for purified nuclear ADP-ribosyl transferase (12). The observed allosteric behavior is likely to arise from the interaction of NAD binding sites either on the same or different enzyme molecules. There is no evidence that nuclear ADP-ribosyl transferase is an oligomeric protein. More than one nuclear ADP-ribosyl transferase molecule is involved however in the... 

As previously shown, 3-fluoro sialosyl fluorides could be employed as donor substrates for TcTS. Due to the fluoro substituent and its pronounced electron-withdrawing features at the anomeric center, these derivatives show delayed reactions with acceptor structures [28]. This effect proved to be useful in determination of essential mechanistic aspects, since the intermediate enzyme substrate complex could be crystallized and studied [25, 26]. By further modification via introduction of a 9-benzamide function as in compound 24 this effect was increased. In contrast to the expected outcome the enhanced binding of this aromatic residue was unexpectedly due to its location in the acceptor-binding site rather than in the glycerol sidechain binding domain, as evidenced by X-ray structural studies (Fig. 11) [52]. 

Introduction of reversible MAO-A inhibitors resulted in significantly improved safety during antidepressant therapy. The potential for hypertensive problems is virtually absent because tyramine is able to displace the dmg from the binding site. Furthermore, selectivity for only MAO-A allows for metabolism with MAO-B. The benzamide derivative moclobemide (Fig. 18.24) was launched in 1990 and represents the most commonly used dmg for this particular purpose. Brofaromine, a benzofiiran derivative, has been found to show very similar properties when compared with moclobemide. It also displayed some promising potential as a weak selective serotonin reuptake inhibitor but clinically studies have been abandoned due to lack of corporate interest. 

PARP inhibitors have been synthesized (11) and are shown in Figs. 11.4 and 11.5. Some of them have been tested in stroke models with good results in terms of preventing infarction (12-18). Most of the inhibitors are based on the structure of nicotinamide and therefore bind to the nicotinamide site of PARP. Several inhibitors are well known, such as nicotinamide, benzamide, and 3-aminobenzamide (19,20). However, new quinazolinone, phen-anthradinone, and other inhibitors are being investigated. Nicotinamide has a of about 5 xM for PARP (19). Activity is decreased in 6-aminonicotinamide, isonicotinamide, 1-methylnicotinamide, 5-methylnicotinamide, 8-methylnicotinamide, and thionicotinamide (Table 11.1 >... 

Substituted benzamides have recently been extensively studied in ternary complexes with LADH-NADH 280-283) in order to deduce and separate hydrophobic, electronic, and steric roles of the substituents for binding. The detailed analyses of these data are compatible with two different binding groups on the amide, one hydrophobic and the other electronic. An attempt has also been made to map the active site from the binding pattern of some aliphatic amides (284) ... 

Benzamides represent a fourth class of HDAC inhibitors. Unlike the other HDAC inhibitors above, benzamides do not conform to the simple pharmacophore model with an obvious metal-binding group connected to a linear spacer. Whether they work by the same mechanism or target an allosteric site on the enzyme is not fully resolved. Nevertheless, they display nanomolar potency, and more than one compound have reached phase I clinical trials for... 

Fig. 16.1.3. Representative antitubulin compounds believed to bind at the benzamide site.

Representative compounds believed to bind at the benzamide site on the basis of evidence from competitive binding or cross-resistance studies (discussed below) are shown in Fig. 16.1.3. Although these compounds differ from zoxamide in their relative toxicity toward different organisms, they appear to bind to the same domain on y -tubulin. Selective toxicity may be governed by structural differences between organisms in this domain. 

Ethaboxam 10, a fungicide currently being developed by LG Life Sciences for the Oomycete market (Fig. 16.1.4), was recently reported to act by disruption of microtubules in Phytophthora infestans [15]. Ethaboxam bears some structural similarity to zarilamide however, it has not yet been established whether ethaboxam binds to tubulin and, if so, whether it binds to the same site as other benzamides. 

Since its first commercial use in 2001, there have been no reports of reduced pathogen sensitivity to zoxamide. Laboratory studies to investigate the potential for resistance development to the benzamide class have been carried out with zoxamide [24], and zarilamide [25], a benzamide that binds to the same site as zoxamide on jS-tubulin [7]. In these studies, attempts to isolate resistant mutants in different Oomycetes using chemical mutagenesis, UV irradiation or adaptation were unsuccessful. These results suggest that the risk for resistance development to zoxamide in its commercial target pathogens is relatively low. 

A total of six crystal structures were obtained and in each case the same connectivity as was observed in the study on tso-nicotinamide was found. For example, the co-crystal of 3-[(benzimidazol-l-yl)methyl]-benzamide with 3-nitrobenzoic acid yielded the expected result. The -OH group of the acid binds to the aromatic nitrogen of the benzimidazole and the amide forms a homomeric dimer (Fig. 4, top). If the system is saturated with acid, a 2 1 co-crystal is obtained with the carboxylic acid interacting with both the benzimidazole site and with the amide moiety (Fig. 4, bottom). The latter structure does offer important additional infor-matimi because it shows that the acid is capable of interacting with both sites so... 

---