Evaluation of the Influence of a Thioether Substituent on the Solid State and Solution Properties of N\u3csub\u3e3\u3c/sub\u3eS-ligated Copper(II) Complexes

Admixture of a N3S(thioether) ligand having two internal hydrogen bond donors (pbnpa: N-2-(phenylthio)ethyl-N,N-bis-((6-neopentylamino-2-pyridyl)methyl)amine; ebnpa: N-2-(ethylthio)ethyl-N,N-bis-((6-neopentylamino-2-pyridyl)methyl)amine) with equimolar amounts of Cu(ClO4)2·6H2O and NaX (X = Cl−, NCO−, or N3−) in CH3OH/H2O yielded the mononuclear Cu(II) derivatives [(pbnpa)Cu–Cl]ClO4 (1), [(ebnpa)Cu–Cl]ClO4 (2), [(pbnpa)Cu–NCO]ClO4 (3), [(ebnpa)Cu–NCO]ClO4 (4), [(pbnpa)Cu–N3]ClO4 (5), and [(ebnpa)Cu–N3]ClO4 (6). Each complex was characterized by FTIR, UV-VIS, EPR, and elemental analysis. Complexes 1, 2, 3 and 6 were characterized by X-ray crystallography. The structural studies revealed that [(pbnpa)Cu–X]ClO4 derivatives (1, 3) exhibit a distorted square pyramidal type geometry, whereas [(ebnpa)Cu–X]ClO4 complexes (2, 6) may be classified as distorted trigonal bipyramidal. EPR studies in CH3OH/CH3CN solution revealed that 1–6 exhibit an axial type spectrum with g∥ \u3e g⊥ \u3e 2.0 and A∥ = 15–17 mT, consistent with a square pyramidal based geometry for the Cu(II) center in each complex. A second species detected in the EPR spectra of 2 and 6 has a smaller A∥ value, consistent with greater spin delocalization on to sulfur, and likely results from geometric distortion of the [(ebnpa)Cu(II)–X]+ ions present in 2 and 6

Inhibition of the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl- ʟ, ʟ-diaminopimelic Acid Desuccinylase from \u3cem\u3eNeisseria meningitidis\u3c/em\u3e by ʟ-Captopril

Binding of the competitive inhibitor ʟ-captopril to the dapE-encoded N-succinyl-ʟ, ʟ-diaminopimelic acid desuccinylase from Neisseria meningitidis (NmDapE) was examined by kinetic, spectroscopic, and crystallographic methods. ʟ-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from Haemophilus influenzae (HiDapE) with an IC50 of 3.3 μM and a measured Ki of 1.8 μM and displayed a dose-responsive antibiotic activity toward Escherichia coli. ʟ-Captopril is also a competitive inhibitor of NmDapE with a Ki of 2.8 μM. To examine the nature of the interaction of ʟ-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co(II)-substituted forms of both HiDapE and NmDapE. EPR and MCD data indicate that the two Co(II) ions in DapE are antiferromagnetically coupled, yielding an S = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of NmDapE in complex with ʟ-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of ʟ-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes

Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation

Genetic Method To Analyze Essential Genes of Escherichia coli▿

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes

Inhibition of the <i>dapE</i>-Encoded <i>N</i>‑Succinyl‑l,l‑diaminopimelic Acid Desuccinylase from <i>Neisseria meningitidis</i> by l‑Captopril

Binding of the competitive inhibitor l-captopril to the <i>dapE</i>-encoded <i>N</i>-succinyl-l,l-diaminopimelic acid desuccinylase from <i>Neisseria meningitidis</i> (<i>Nm</i>DapE) was examined by kinetic, spectroscopic, and crystallographic methods. l-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from <i>Haemophilus influenzae</i> (<i>Hi</i>DapE) with an IC₅₀ of 3.3 μM and a measured <i>K</i>_i of 1.8 μM and displayed a dose-responsive antibiotic activity toward <i>Escherichia coli</i>. l-Captopril is also a competitive inhibitor of <i>Nm</i>DapE with a <i>K</i>_i of 2.8 μM. To examine the nature of the interaction of l-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co­(II)-substituted forms of both <i>Hi</i>DapE and <i>Nm</i>DapE. EPR and MCD data indicate that the two Co­(II) ions in DapE are antiferromagnetically coupled, yielding an <i>S</i> = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of <i>Nm</i>DapE in complex with l-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of l-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes