Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study.

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8s, and permits the measurement of up to 10,000 samples per day. Concentration-response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R(2) = 0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi-PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi-PFK are presented, and the ways in which SPE-MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.

Mismatch repair proteins collaborate with methyltransferases in the repair of O(6)-methylguanine.

DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O(6)-methylguanine (O(6)mG), which stably pairs with thymine during replication and thereby creates a promutagenic O(6)mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O(6)mG:T mismatches can lead to cell death or result in G:C-->A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O(6)mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O(6)mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O(6)mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O(6)mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O(6)mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O(6)mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.

Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity.

Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development.

Advances in label-free screening approaches for studying histone acetyltransferases.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all possible acetylation states of a peptide concurrently to measure HAT activity. The second approach measures acetylation reactions, on both peptides and whole protein substrates, via direct detection of the acetyl-coenzyme A cosubstrate and coenzyme A coproduct. Lastly, the authors demonstrate the ability to monitor directly the acetylation state of whole histone proteins in the same high-throughput manner using time-of-flight mass spectrometry. The generation of compound-mediated inhibition data using each of these techniques establishes mass spectrometry as a versatile, label-free, and biologically relevant screening approach to this challenging target class.

Advances in label-free screening approaches for studying sirtuin-mediated deacetylation.

The sirtuin enzymes, a class of NAD(+)-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were applied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data, including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via O-acetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of whole-histone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins using more biologically relevant molecules.

A bifunctional platinum(II) antitumor agent that forms DNA adducts with affinity for the estrogen receptor.

A strategy is described for the re-design of DNA damaging platinum(II) complexes to afford elevated toxicity towards cancer cells expressing the estrogen receptor (ER). Two platinum-based toxicants are described in which a DNA damaging warhead, [Pt(en)Cl(2)] (en, ethylenediamine), is tethered to either of two functional groups. The first agent, [6-(2-amino-ethylamino)-hexyl]-carbamic acid 2-[6-(7alpha-estra-1,3,5,(10)-triene)-hexylamino]-ethyl ester platinum(II) dichloride ((Est-en)PtCl(2)), terminates in a ligand for the ER. The second agent is a control compound lacking the steroid; this compound, N-[6-(2-amino-ethylamino)-hexyl]-benzamide platinum(II) dichloride ((Bz-en)PtCl(2))), terminates in a benzamide moiety, which lacks affinity for the ER. Using a competitive binding assay, Est-en had 28% relative binding affinity (RBA) for the ER as compared to 17beta-estradiol. After covalent binding to a synthetic DNA duplex 16-mer, the compound retained its affinity for the ER; specificity of the binding event was demonstrated by the ability of free 17beta-estradiol as a competitor to disrupt the DNA adduct-ER complex. The (Est-en)PtCl(2) compound showed higher toxicity against the ER positive ovarian cancer cell line CAOV3 than did the control compound. (Est-en)PtCl(2) was also more toxic to the ER positive breast cancer line, MCF-7, than to an ER negative line, MDA-MB231.

A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells.

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.