Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

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.

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-ß-lactamase.

BACKGROUND: Metallo-ß-lactamases (MBLs) play an important role in the emergence of microbial resistance to ß-lactam antibiotics, and are hence considered targets for the design of novel therapeutics. We here report on the inhibitory effect of peptides containing multiple arginine residues on VIM-2, a clinically important MBL from Pseudomonas aeruginosa. METHODS: Enzyme kinetic assays in combination with fluorescence spectroscopy and stopped-flow UV-Vis spectrophotometry were utilized to explore the structure-activity relationship of peptides as inhibitors of VIM-2. RESULTS: Our studies show that the inhibitory potency of the investigated peptides was mainly dependent on the number of arginine residues in the center of the peptide sequence, and on the composition of the N-terminus. The most potent inhibitors were found to curtail enzyme function in the mid-to-low nanomolar range. Salts generally reduced peptide-mediated inhibition. Analysis of the mode of inhibition suggests the peptides to act as mixed-type inhibitors with a higher affinity for the enzyme-substrate complex. Stopped-flow UV-Vis and fluorescence studies revealed the peptides to induce rapid protein aggregation, a phenomenon strongly correlated to the peptides' inhibitory potency. Inhibition of IMP-1 (another subclass B1 MBL) by the peptides was found to be much weaker than that observed with VIM-2, a finding which might be related to subtle molecular differences in the protein surfaces. CONCLUSION: The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs. GENERAL SIGNIFICANCE: Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.

PLCß1a and PLCß1b selective regulation and cyclin D3 modulation reduced by kinamycin F during k562 cell differentiation.

Here we report that both PLCß1a and PLCß1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction of both PLCß1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCß1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCß1b induced an increase in γ-globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCß1 signaling pathway. Namely the amplification of the expression of the PLCß1a, but not of PLCß1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCß1b is mainly present in the nucleus in respect to PLCß1a. Our data indicate that the amplification of PLCß1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis.

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.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as ß-lactamase substrates.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of ß-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of ß-lactamases. Kinetic parameters were determined with eight different ß-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by ß-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

Evaluation of four microbial Class II fructose 1,6-bisphosphate aldolase enzymes for use as biocatalysts.

Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms.

Cyclobutanone analogues of beta-lactams revisited: insights into conformational requirements for inhibition of serine- and metallo-beta-lactamases.

The most important mode of bacterial resistance to beta-lactam antibiotics is the expression of beta-lactamases. New cyclobutanone analogues of penams and penems have been prepared and evaluated for inhibition of class A, B, C, and D beta-lactamases. Inhibitors which favor conformations in which the C4 carboxylate is equatorial were found to be more potent than those in which the carboxylate is axial, and molecular modeling studies with enzyme-inhibitor complexes indicate that an equatorial orientation of the carboxylate is required for binding to beta-lactamases. An X-ray structure of OXA-10 complexed with a cyclobutanone confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. An unsaturated penem analogue was also found to enhance the potency of meropenem against carbapenem-resistant MBL-producing strains of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia. These cyclobutanones represent the first type of reversible inhibitors to show moderate (low micromolar) inhibition of both serine- and metallo-beta-lactamases and should be considered for further development into practical inhibitors.

A biogenetically-inspired synthesis of a ring-D model of kinamycin F: insights into the conformation of ring D.

An efficient three-step construction of the highly oxygenated D-ring of the kinamycin antibiotics is reported for a simple model system. A comparison of the spectroscopic characteristics of the synthetic models with those of natural kinamycin F, which is suspected to be the bioactive form of the kinamycins, leads to the conclusion that the favored D-ring conformation of kinamycin F differs from that of the other partially or fully acylated variants.

Cyclobutanone mimics of penicillins: effects of substitution on conformation and hemiketal stability.

The tendency for carbocyclic analogues of penicillins to undergo hydrate and hemiketal formation is central to their ability to function as beta-lactamase inhibitors. 2-Thiabicyclo[3.2.0]heptan-6-one-4-carboxylates with alkoxy functionality at C3 have been prepared through two complementary diastereoselective substitution reactions following a highly stereoselective chlorination with sulfuryl chloride. We have found that carbocyclic analogues with 3beta substituents favor an endo envelope conformation in solution, the solid state, and the gas phase, whereas those with 3alpha substituents adopt an exo envelope. Evidence from X-ray crystal structures and ab initio calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favors the C3 substituent in an axial orientation. In addition, the envelope conformation of the bicycle, which is determined by the stereochemistry of the C3 substituent, has a dramatic effect on the ability of the cyclobutanone to undergo hemiketal formation in methanol-d4.

Assays for beta-lactamase activity and inhibition.

The ability, either innate or acquired, to produce beta-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in beta-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of beta-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn2+-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of beta-lactam antibiotics in clinical medicine. This chapter concerns procedures for the evaluation of the catalytic activity of beta-lactamases as a means to screen compounds for their inhibitory potency.

Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F.

The bacterial metabolite kinamycin F, which is being investigated as a potent antitumor agent, contains an unusual and potentially reactive diazo group, a paraquinone, and a phenol functional group. Kinamycin F reacted with glutathione (GSH) in a complex series of reactions which suggested that kinamycin F may have its cytotoxicity modulated by GSH. Consistent with this idea, 2-oxo-4-thiazolidinecarboxylic acid treatment to increase cellular GSH levels and buthionine sulfoximine treatment to decrease GSH levels resulted in decreased and increased kinamycin F cytotoxicity, respectively, in K562 leukemia cells. Kinamycin F weakly bound to DNA and induced DNA damage in K562 cells that was independent of GSH levels. The GSH-promoted DNA nicking induced by kinamycin F in vitro was attenuated by deferoxamine, dimethyl sulfoxide, and catalase, which indicated that DNA damage initiated by this agent occurred in an iron-, hydrogen-peroxide-, and hydroxyl-radical-dependent manner. Electron paramagnetic resonance spectroscopy experiments showed that the GSH/kinamycin F system produced a semiquinone free radical and that the hydrogen peroxide/peroxidase/kinamycin F system generated a phenoxyl free radical. In conclusion, the results indicated that kinamycin F cytotoxicity may be due to reductive and/or peroxidative activation to produce DNA-and protein-damaging species.

Total synthesis of isoprekinamycin: structural evidence for enhanced diazonium ion character and growth inhibitory activity toward cancer cells.

The structurally novel diazobenzo[a]fluorene antibiotic isoprekinamycin (IPK) has been synthesized for the first time employing a Suzuki coupling of a brominated AB ring synthon with a boronate ester representing the D ring, followed by anionic cyclization and appropriate functional group manipulations. The first indication that the diazobenzo[a]fluorene system exhibits in vitro anticancer activity is provided and X-ray crystallographic evidence for enhancement of diazonium ion character as a consequence of intramolecular H-bonding is described.

68Zn isotope exchange experiments reveal an unusual kinetic lability of the metal ions in the di-zinc form of IMP-1 metallo-beta-lactamase.

The apparently paradoxical behaviour of facile exchange (kinetic lability) of tightly bound (thermodynamic stability) zinc ions in the enzyme IMP-1 metallo-beta-lactamase with Zn-68 and cadmium ions, as indicated by in-torch vaporization inductively-coupled plasma mass spectrometry (ITV-ICP-MS) and electrospray-ionization mass spectrometry (ESI-MS), is consistent with the involvement of a third metal ion in promoting Lewis acid/base type exchange processes.

IMP-1 metallo-beta-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry.

Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme. Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions. This mononuclear Zn species was found to be catalytically competent. Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).

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.

Kinamycin F downregulates cyclin D3 in human leukemia K562 cells.

The bacterial metabolite kinamycin F, which contains an unusual and potentially reactive diazo group, is being investigated as an antitumor agent with a potentially novel target. Treatment of K562 cells with kinamycin F induced erythroid differentiation, a rapid apoptotic response, induction of caspase-3/7 activities and a delayed cell cycle progression through the S and G(2)/M phases. Kinamycin F caused a selective reduction of cyclin D3 protein, which appeared to be mediated at the level of transcription, rather than by affecting the stability of either cyclin D3 protein or mRNA. Thus cyclin D3 is a potential target of kinamycin F.