Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis.

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo ß-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.

Cephalosporins inhibit human metallo ß-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo.

Bacterial metallo-ß-lactamases (MBLs) are involved in resistance to ß-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact ß-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.

The Chemical Biology of Human Metallo-ß-Lactamase Fold Proteins.

The αßßα metallo ß-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all ß-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

Crystal structure of human persulfide dioxygenase: structural basis of ethylmalonic encephalopathy.

The ethylmalonic encephalopathy protein 1 (ETHE1) catalyses the oxygen-dependent oxidation of glutathione persulfide (GSSH) to give persulfite and glutathione. Mutations to the hETHE1 gene compromise sulfide metabolism leading to the genetic disease ethylmalonic encephalopathy. hETHE1 is a mono-iron binding member of the metallo-ß-lactamase (MBL) fold superfamily. We report crystallographic analysis of hETHE1 in complex with iron to 2.6 Å resolution. hETHE1 contains an αßßα MBL-fold, which supports metal-binding by the side chains of an aspartate and two histidine residues; three water molecules complete octahedral coordination of the iron. The iron binding hETHE1 enzyme is related to the 'classical' di-zinc binding MBL hydrolases involved in antibiotic resistance, but has distinctive features. The histidine and aspartate residues involved in iron-binding in ETHE1, occupy similar positions to those observed across both the zinc 1 and zinc 2 binding sites in classical MBLs. The active site of hETHE1 is very similar to an ETHE1-like enzyme from Arabidopsis thaliana (60% sequence identity). A channel leading to the active site is sufficiently large to accommodate a GSSH substrate. Some of the observed hETHE1 clinical mutations cluster in the active site region. The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors.

Bisthiazolidines: A Substrate-Mimicking Scaffold as an Inhibitor of the NDM-1 Carbapenemase.

Pathogenic Gram-negative bacteria resistant to almost all ß-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-ß-lactamases (MBLs) produced by these bacteria inactivate most ß-lactam antibiotics, including the carbapenems, which are "last line therapies" for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of ß-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with ß-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 µM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.

Rhodanine hydrolysis leads to potent thioenolate mediated metallo-ß-lactamase inhibition.

The use of ß-lactam antibiotics is compromised by resistance, which is provided by ß-lactamases belonging to both metallo (MBL)- and serine (SBL)-ß-lactamase subfamilies. The rhodanines are one of very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in ß-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including (19)F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.

The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering.

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1ß in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1ß secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1ß but not the biosynthesis of pro-IL-1ß, indicating that redox remodeling is responsible for IL-1ß processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1ß processing and secretion. High doses (5 mM) of H(2)O(2) overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1ß secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1ß secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.

Defective repair of 5-hydroxy-2'-deoxycytidine in Cockayne syndrome cells and its complementation by Escherichia coli formamidopyrimidine DNA glycosylase and endonuclease III.

Repair of the oxidized purine 8-oxo-7,8-dihydro-2'-deoxyguanosine is inefficient in cells belonging to both complementation groups A and B of Cockayne syndrome (CS), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that both CS-A and CS-B cells are also defective in the repair of 5-hydroxy-2'-deoxycytidine (5-OHdC), an oxidized pyrimidine with cytotoxic and mutagenic properties. The defect in the repair of oxidatively damaged DNA in CS cells thus extends to oxidized pyrimidines, indicating a general flaw in the repair of oxidized lesions in this syndrome. The defect could not be reproduced in in vitro repair experiments on oligonucleotide substrates, suggesting a role for both CS-A and CS-B proteins in chromatin remodeling during 5-OHdC repair. Expression of Escherichia coli formamidopyrimidine DNA glycosylase (FPG) or endonuclease III complemented the 5-OHdC repair deficiency. Hence, the expression of a single enzyme, FPG from E. coli, stably corrects the delayed removal of both oxidized purines and oxidized pyrimidines in CS cells.