A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.

Concise synthesis of didebromohamacanthin A and demethylaplysinopsine: addition of ethylenediamine and guanidine derivatives to the pyrrole-amino acid diketopiperazines in oxidative conditions.

Oxidative nucleophilic addition of ethylenediamine and guanidine derivatives to pyrrole-amino acid diketopiperazines was shown to provide substituted 5,6-dihydro-2(1H)-piperazinones, quinoxalinones, and 2-aminoimidazolones. On the basis of this methodology, a concise approach to natural products didebromohamacanthin A and demethylaplysinopsine has been demonstrated.

Chromophore-linked substrate (CLS405): probing metallo-ß-lactamase activity and inhibition.

Serine- and metallo-ß-lactamases present a threat to the clinical use of nearly all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Efforts to develop metallo-ß-lactamase (MBL) inhibitors require suitable screening platforms to allow the rapid determination of ß-lactamase activity and efficient inhibition. Unfortunately, the platforms currently available are not ideal for this purpose. Further progress in MBL inhibitor identification requires inexpensive and widely applicable assays. Herein the identification of an inexpensive and stable chromogenic substrate suitable for use in assays of clinically relevant MBLs is described. (6R,7R)-3-((4-Nitrophenoxy)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (CLS405) was synthesised in a three-step protocol. CLS405 was then characterised spectroscopically, and its stability and kinetic properties evaluated. With a Δλmax value of 100 nm between the parent and hydrolysis product, a higher analytical accuracy is possible with CLS405 than with commonly used chromogenic substrates. The use of CLS405 in assays was validated by MBL activity measurements and inhibitor screening that resulted in the identification of N-hydroxythiazoles as new inhibitor scaffolds for MBLs. Further evaluation of the identified N-hydroxythiazoles against a panel of clinically relevant MBLs showed that they possess inhibitory activities in the mid- to low-micromolar range. The findings of this study provide both a useful tool compound for further inhibitor identification, and novel scaffolds for the design of improved MBL inhibitors with potential as antibiotics against resistant strains of bacteria.

Unprecedented biomimetic homodimerization of oroidin and clathrodin marine metabolites in the presence of HMPA or phosphonate salt tweezers.

The first biomimetic homodimerization of oroidin and clathrodin was effected in the presence HMPA and diphosphonate salts, strong guanidinium and amide chelating agents. The intermolecular associations probably interfere with the entropically and kinetically favored intramolecular cyclizations. Use of oroidin·(1)/2HCl salt or clathrodin·(1)/2HCl was indicative in the presence of the ambident nucleophilic and electrophilic tautomers of the 2-aminoimidazolic oroidin and clathrodin precursors. Surprisingly, the homodimerization of oroidin led to the nagelamide D skeleton, while the homodimerization of clathrodin gave the benzene para-symmetrical structure 19. The common process was rationalized from tautomeric precursors I and III.

Structural basis for inhibition of the fat mass and obesity associated protein (FTO).

The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry- and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.

1,3,5-Trisubstituted benzenes as fluorescent photoaffinity probes for human carbonic anhydrase II capture.

The synthesis of small molecule based 1,3,5-trisubstituted benzenes for photo-mediated capture of human carbonic anhydrase II with visualisation by fluorescence is described.

5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation.

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

Reporter ligand NMR screening method for 2-oxoglutarate oxygenase inhibitors.

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and K(D) values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry.

Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases.

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Studies of silicophosphate derivatives by 31P-->29Si CP MAS NMR.

We show that it is possible to efficiently transfer magnetization from 31P to 29Si, using variable amplitude CP MAS experiment. This experiment is demonstrated by using Si5O(PO4)6, the synthesis protocol of which is described. From the obtained results, we show that the experiment allows the spectral edition of 29Si spectra from 31P-->29Si CP, enabling the studies of derivatives involving Si-O-P linkages, such as phosphosilicate glasses, microporous silicoaluminophosphates (SAPO) and bioactive phosphosilicates.