Characterization of 3-[(Carboxymethyl)thio]picolinic Acid: A Novel Inhibitor of Phosphoenolpyruvate Carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) has traditionally been characterized for its role in the first committed step of gluconeogenesis. The current understanding of PEPCK's metabolic role has recently expanded to include it serving as a general mediator of tricarboxylic acid cycle flux. Selective inhibition of PEPCK in vivo and in vitro has been achieved with 3-mercaptopicolinic acid (MPA) (Ki ∼ 8 µM), whose mechanism of inhibition has been elucidated only recently. On the basis of crystallographic and mechanistic data of various inhibitors of PEPCK, MPA was used as the initial chemical scaffold to create a potentially more selective inhibitor, 3-[(carboxymethyl)thio]picolinic acid (CMP), which has been characterized both structurally and kinetically here. These data demonstrate that CMP acts as a competitive inhibitor at the OAA/PEP binding site, with its picolinic acid moiety coordinating directly with the M1 metal in the active site (Ki ∼ 29-55 µM). The extended carboxy tail occupies a secondary binding cleft that was previously shown could be occupied by sulfoacetate (Ki ∼ 82 µM) and for the first time demonstrates the simultaneous occupation of both OAA/PEP subsites by a single molecular structure. By occupying both the OAA/PEP binding subsites simultaneously, CMP and similar molecules can potentially be used as a starting point for the creation of additional selective inhibitors of PEPCK.

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates.

There are currently no clinically available inhibitors of metallo-ß-lactamases (MBLs), enzymes that hydrolyze ß-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 µM; Ki values of 0.03-1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of ß-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Cyclobutanone Mimics of Intermediates in Metallo-ß-Lactamase Catalysis.

The most important resistance mechanism to ß-lactam antibiotics involves hydrolysis by two ß-lactamase categories: the nucleophilic serine and the metallo-ß-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable ß-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19 F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13 C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all ß-lactamase classes.

Experimental estimation of average fidelity of a Clifford gate on a 7-qubit quantum processor.

One of the major experimental achievements in the past decades is the ability to control quantum systems to high levels of precision. To quantify the level of control we need to characterize the dynamical evolution. Full characterization via quantum process tomography is impractical and often unnecessary. For most practical purposes, it is enough to estimate more general quantities such as the average fidelity. Here we use a unitary 2-design and twirling protocol for efficiently estimating the average fidelity of Clifford gates, to certify a 7-qubit entangling gate in a nuclear magnetic resonance quantum processor. Compared with more than 10^{8} experiments required by full process tomography, we conducted 1656 experiments to satisfy a statistical confidence level of 99%. The average fidelity of this Clifford gate in experiment is 55.1%, and rises to at least 87.5% if the signal's decay due to decoherence is taken into account. The entire protocol of certifying Clifford gates is efficient and scalable, and can easily be extended to any general quantum information processor with minor modifications.

Development of metal-chelating inhibitors for the Class II fructose 1,6-bisphosphate (FBP) aldolase.

It has long been suggested that the essential and ubiquitous enzyme fructose 1,6-bisphosphate (FBP) aldolase could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependant (Class II) FBP aldolase, as opposed to higher organisms which possess a Schiff-base forming (Class I) FBP aldolase. We have tested the capacity of derivatives of the metal-chelating compound dipicolinic acid (DPA), as well a thiol-containing compound, to inhibit purified recombinant Class II FBP aldolases from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, Bacillus anthracis, and from the Rice Blast causative agent Magnaporthe grisea. The aldolase from M. tuberculosis was the most sensitive to the metal-chelating inhibitors, with an IC(50) of 5.2 µM with 2,3-dimercaptopropanesulfonate (DMPS) and 28 µM with DPA. DMPS and the synthesized inhibitor 6-(phosphonomethyl)picolinic acid inhibited the enzyme in a time-dependent, competitive fashion, with second order rate constants of 273 and 270 M(-1) s(-1) respectively for the binding of these compounds to the M. tuberculosis aldolase's active site in the presence of the substrate FBP (K(M) 27.9 µM). The most potent first generation inhibitors were modeled into the active site of the M. tuberculosis aldolase structure, with results indicating that the metal chelators tested cannot bind the catalytic zinc in a bidentate fashion while it remains in its catalytic location, and that most enzyme-ligand interactions involve the phosphate binding pocket residues.