Laura Marrone

N-Arylsulfonyl Hydrazones as Inhibitors of IMP-1 Metallo-β-Lactamase

ABSTRACT Members of a family of N-arylsulfonyl hydrazones have been identified as novel inhibitors of IMP-1, a metallo-β-lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efficient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo-β-lactamase activity exhibited by this class of compounds.

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.

Lysine: N6-Hydroxylase: Stability and Interaction with Ligands

Recombinant lysine:N6-hydroxylase, rIucD, which is isolated as an apoenzyme, requires FAD and NADPH for its catalytic function. rIucD preparations have been found to undergo time-dependent loss in monooxygenase function due to aggregation from the initial tetrameric state to a polytetrameric form(s), a process which is reversible by treatment with thiols. Ligand-in-duced conformational changes in rIucD were assessed by monitoring its CD spectra, DSC profile, and susceptibility to both endo- as well as exopeptidases. The first two methods indicated the absence of any significant conformational change in rIucD, while the last approach revealed that FAD, and its analog ADP, can protect the protein from the deleterious action of proteases. NADPH was partially effective and L-lysine was ineffective in this regard. Deletion of the C-terminal segment, either by treatment with carboxypeptidase Y or by mutagenesis of iucD, results in the loss of rIucDs monooxygenase activity. These findings demonstrate the crucial role of the C-terminal segment in maintaining rIucD in its native conformation.

Effect of selective cysteine --> alanine replacements on the catalytic functions of lysine: N6-hydroxylase.

Recombinant lysine: N6-hydroxylase, rIucD, catalyzes the conversion of L-lysine to its N6-hydroxy derivative. Re-examination of the nucleotide sequence of iucD, the gene encoding for the enzyme, has revealed a few discrepancies in the data documented in literature and the corrected version is presented. The revised nucleotide sequence predicts the presence of five cysteine residues in the primary structure of IucD. Two of these residues, cysteine 51 and cysteine 158 are alkylatable by iodoacetate in the native conformation of the protein resulting in a loss of monooxygenase activity while their replacement with alanine has no such adverse effect. Site directed mutagenesis studies have enabled an assessment of the reactivity of these cysteine residue(s) towards thiol modifying agents.

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

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of β-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of β-lactamases. Kinetic parameters were determined with eight different β-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 β-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

Assays for Β-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.

Cofactor- and substrate-binding domains in flavin-dependent N-hydroxylating enzymes

A recent article in TiBS, by Macheroux and co-workers[1xStehr, M. et al. Trends Biochem. Sci. 1998; 23: 56–57Abstract | Full Text PDF | PubMed | Scopus (31)See all References[1], pertains to the cofactor- and substrate-binding domains in flavin-dependent N-hydroxylating enzymes. We would like to restrict our comments primarily to lysine-N6-hydroxylase, IucD, an enzyme that we have been investigating for some years.Studies of IucD–PhoA and IucD–LacZ fusion proteins[2xHerrero, M., de Lorenzo, V., and Neilands, J.B. J. Bacteriol. 1988; 170: 56–64PubMedSee all References[2]support the observation that IucD is normally membrane bound[3xGoh, C.J., Szczepan, E.W., Menhart, N., and Viswantha, T. Biochim. Biophys. Acta. 1989; 990: 240–245Crossref | PubMedSee all References[3]. These studies suggest that at least two domains in IucD attach to the cytoplasmic side of the plasma membrane. The first membrane-attachment domain appears to reside in the first 25 residues of the protein, and its sequence resembles that of the signal peptide[2xHerrero, M., de Lorenzo, V., and Neilands, J.B. J. Bacteriol. 1988; 170: 56–64PubMedSee all References[2]. Although Diekmann and co-workers[4xPlattner, H.J. et al. Biol. Metals. 1989; 2: 1–5Crossref | PubMed | Scopus (13)See all References, 5xMacheroux, P., Plattner, H.J., Romaguera, A., and Diekmann, H. Eur. J. Biochem. 1993; 213: 995–1002Crossref | PubMed | Scopus (15)See all References]have described a soluble form of IucD[4xPlattner, H.J. et al. Biol. Metals. 1989; 2: 1–5Crossref | PubMed | Scopus (13)See all References, 5xMacheroux, P., Plattner, H.J., Romaguera, A., and Diekmann, H. Eur. J. Biochem. 1993; 213: 995–1002Crossref | PubMed | Scopus (15)See all References], an inability to isolate membrane-free preparations of the enzyme prompted us to develop recombinant, cytoplasmic forms of the protein by using strategies based on the gene-fusion approach[6xThariath, A.M., Socha, D., Valvano, M.A., and Viswanatha, T. J. Bacteriol. 1993; 175: 589–596PubMedSee all References[6].We contructed three different plasmids, pAT2, pAT3 and pAT5, which encode modified IucD forms (rIucDs) that possess altered N-termini[6xThariath, A.M., Socha, D., Valvano, M.A., and Viswanatha, T. J. Bacteriol. 1993; 175: 589–596PubMedSee all References[6]. Escherichia coli cells transformed with pAT5 and pAT3 produced cytoplasmic forms of lysine-N6-hydroxylase[6xThariath, A.M., Socha, D., Valvano, M.A., and Viswanatha, T. J. Bacteriol. 1993; 175: 589–596PubMedSee all References, 7xThariath, A.M., Fatum, K.L., Valvano, M.A., and Viswanatha, T. Biochim. Biophys. Acta. 1993; 1203: 27–35Crossref | PubMed | Scopus (17)See all References, 8xSee all References]. The rIucD encoded by pAT2 can be produced either as a membrane-associated form (rIucD 426) or as a cytoplasmic form (rIucD 456)—production of the latter form being dependent on expression in a supK strain of E. coli[6xThariath, A.M., Socha, D., Valvano, M.A., and Viswanatha, T. J. Bacteriol. 1993; 175: 589–596PubMedSee all References[6]. The incorporation of the desired fusion and the absence of any other mutations in the IucD component were confirmed by determination of the nucleotide sequence of each construct. There were a few discrepancies relative to the previously reported nucleotide sequence of IucD[2xHerrero, M., de Lorenzo, V., and Neilands, J.B. J. Bacteriol. 1988; 170: 56–64PubMedSee all References[2]; however, these are consistent with the revised sequence that has recently been published[9xMarrone, L. and Viswanatha, T. Biochim. Biophys. Acta. 1997; 1343: 263–277Crossref | PubMed | Scopus (8)See all References[9].Our rIucD 398 construct contains 19 amino acid residues derived from β-galactosidase; the rest of the fusion protein comprises residues Val48–Thr426 of wild-type IucD. Thus, rIucD 398—which is catalytically active—lacks the N-terminal segment that Macheroux and co-workers[1xStehr, M. et al. Trends Biochem. Sci. 1998; 23: 56–57Abstract | Full Text PDF | PubMed | Scopus (31)See all References[1]identified as the FAD-binding domain. We therefore think that it would be prudent to defer assignment of specific cofactor- and substrate-binding domains until a thorough characterization based on the elucidation of three-dimensional structure of the protein has been achieved. The N5-ornithine hydroxylases PvdA and Sid1 remain to be characterized. Such a paucity of information calls for caution to be exercised in the assignment of specific functional domains in this protein.

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

There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes that hydrolyze β-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 β-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.