Structural basis for different substrate profiles of two closely related class D β-lactamases and their inhibition by halogens

OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site β5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 Å resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 Å resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the β5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme

Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity

Engineering Specificity from Broad to Narrow: Design of a β‑Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β‑Lactamase

The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (<i>K</i>_i = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (<i>K</i>_i = 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 β-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a <i>K</i>_i of 0.4 nM, while drastically losing affinity for TEM-1 with a <i>K</i>_i > 10 μM. The K74T:W112D mutant therefore binds KPC-2 β-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (<i>K</i>_i > 10 μM) for other class A β-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 β-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 β-lactamase in lysates from clinical bacterial isolates containing multiple β-lactamases

Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

<div><p>Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.</p></div

Verrucarin A increases cancer cell chemosensitivity to other anti-cancer drugs.

<p>(A–D) A549 cells were treated with verrucarin A in combination with gefitinib, BEZ235, gemcitabine, or docetaxel. (E) T-47D cells were treated with verrucarin A in combination with tamoxifen. (F) MDA-MB-231 cells were treated with verrucarin A in combination with paclitaxel. All cells were treated for 72 h, followed by MTS assay.</p

Verrucarin A selectively reduces SRC-3 protein levels while it does not reduce CARM-1 and p300 protein levels.

<p>(A-B) A549 cells were treated with verrucarin A at different concentrations (0, 10, 20, 50, 100, and 200 nM) for 24 h, then Western analysis was performed to quantitate SRC-1, SRC-2, SRC-3, CARM1, and p300 proteins.</p