The Chemical Biology of Human Metallo-ß-Lactamase Fold Proteins.

The αßßα metallo ß-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all ß-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

A hydroxamic-acid-containing nucleoside inhibits DNA repair nuclease SNM1A.

Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-ß-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-ß-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Structural Basis of Metallo-ß-Lactamase Inhibition by Captopril Stereoisomers.

ß-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-ß-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all ß-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-ß-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Role of Telomeres and Telomeric Proteins in Human Malignancies and Their Therapeutic Potential.

Telomeres are the ends of linear chromosomes comprised of repetitive nucleotide sequences in humans. Telomeres preserve chromosomal stability and genomic integrity. Telomere length shortens with every cell division in somatic cells, eventually resulting in replicative senescence once telomere length becomes critically short. Telomere shortening can be overcome by telomerase enzyme activity that is undetectable in somatic cells, while being active in germline cells, stem cells, and immune cells. Telomeres are bound by a shelterin complex that regulates telomere lengthening as well as protects them from being identified as DNA damage sites. Telomeres are transcribed by RNA polymerase II, and generate a long noncoding RNA called telomeric repeat-containing RNA (TERRA), which plays a key role in regulating subtelomeric gene expression. Replicative immortality and genome instability are hallmarks of cancer and to attain them cancer cells exploit telomere maintenance and telomere protection mechanisms. Thus, understanding the role of telomeres and their associated proteins in cancer initiation, progression and treatment is very important. The present review highlights the critical role of various telomeric components with recently established functions in cancer. Further, current strategies to target various telomeric components including human telomerase reverse transcriptase (hTERT) as a therapeutic approach in human malignancies are discussed.

Squaramide-Based 5'-Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A.

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N-oxyamide and squaramide modifications at the 5'-end of oligonucleotides as phosphate replacements and their biological evaluation using the 5'-exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5'-phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid-phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5'-phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5'-terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

In Silico Fragment-Based Design Identifies Subfamily B1 Metallo-ß-lactamase Inhibitors.

Zinc ion-dependent ß-lactamases (MBLs) catalyze the hydrolysis of almost all ß-lactam antibiotics and resist the action of clinically available ß-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-ß-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.

Exploring staff perceptions on the role of physical environment in dementia care setting.

This study explored staff perceptions of the role of physical environment in dementia care facilities in affecting resident's behaviors and staff care practice. We conducted focus groups with staff (n = 15) in two purposely selected care facilities in Vancouver, Canada. Focus group participants included nurses, care aides, recreation staff, administrative staff, and family. Data analysis revealed two themes: (a) a supportive physical environment contributes positively to both quality of staff care interaction and residents' quality of life and (b) an unsupportive physical environment contributes negatively to residents' quality of life and thereby makes the work of staff more challenging. The staff participants collectively viewed that comfort, familiarity, and an organized space were important therapeutic resources for supporting the well-being of residents. Certain behaviors of residents were influenced by poor environmental factors, including stimulation overload, safety risks, wayfinding challenge, and rushed care This study demonstrates the complex interrelationships among the dementia care setting's physical environment, staff experiences, and residents' quality of life.

Cephalosporins inhibit human metallo ß-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo.

Bacterial metallo-ß-lactamases (MBLs) are involved in resistance to ß-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact ß-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.