Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

Objectives: We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. Methods: We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Results: Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. Conclusions: This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Novel Peptide Therapeutic Approaches for Cancer Treatment.

Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.

Product inhibition kinetics determinations - Substrate interaction affinity and enzymatic kinetics using one quantitative FRET assay.

Product inhibition is a common phenomenon during enzyme-catalyzed reactions. Almost all product molecules of an enzyme reaction should have some structural similarities to the substrate, and can thus still have affinities to the active site of the enzyme as product inhibitor. Currently, the characterizations of product inhibition are generally carried out by different methods to determine product binding affinity to the enzyme and the enzyme kinetics parameters, and then these parameters are combined to determine product inhibition. However, due to different sensitivity and variations, kinetics parameters determined from different methods are often not compatible, resulting in not accurate measurement. Here, we report a novel method that determines the two different classes of kinetics parameters, IC50 and Ki(or KD), Kcat and KM, using one single assay method-quantitative FRET(qFRET) assay for characterizing the product inhibition of pre-SUMO1's maturation by its protease SENP1. One method to determine all kinetics parameters provides, for the first time, not only a convenient method to determine all kinetics parameters, but more importantly, a novel approach to combine different measurements with mutually compatible results and errors.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Human ARMT1 structure and substrate specificity indicates that it is a DUF89 family damage-control phosphatase.

Metabolite damage control is a critical but poorly defined aspect of cellular biochemistry, which likely involves many of the so far functionally uncharacterized protein domain (domains of unknown function; DUFs). We have determined the crystal structure of the human DUF89 protein product of the C6ORF211 gene to 1.85 Å. The crystal structure shows that the protein contains a core α-ß-α fold with an active site-bound metal ion and α-helical bundle N-terminal cap, which are both conserved features of subfamily III DUF89 domains. The biochemical activities of the human protein are conserved with those of a previously characterized budding yeast homolog, where an in vitro phosphatase activity is supported by divalent cations that include Co2+, Ni2+, Mn2+ or Mg2+. Full steady-state kinetics parameters of human DUF89 using a standard PNPP phosphatase assay revealed a six times higher catalytic efficiency in presence of Co2+ compared to Mg2+. The human enzyme targets a number of phosphate substrates similar to the budding yeast homolog, while it lacks a previously indicated methyltransferase activity. The highest activity on substrate was observed with fructose-1-phosphate, a potent glycating agent, and thus human DUF89 phosphatase activity may also play a role in limiting the buildup of phospho-glycan species and their related damaged metabolites.

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.

Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS.

Earlier studies from our laboratory have demonstrated that Oxyresveratrol (OXY), a hydroxyl-substituted stilbene, exhibits potent inhibition of human melanoma cell proliferation. The present study defines a cytotoxic effect of OXY on the highly chemo-resistant, triple-negative human breast cancer cell line MDA-MB-231. OXY-mediated cell death resulted in accumulation of cells at the sub-G1 phase of the cell cycle, induced chromatin condensation, DNA fragmentation, phosphatidylserine externalization and PARP cleavage, indicative of apoptosis. Interestingly, morphology and cell viability studies with the pan-caspase inhibitor, QVD-OPH revealed that OXY-induced cell death was caspase-independent. Docking studies also showed that OXY can bind to the S1 site of caspase-3, and could also exert an inhibitory effect on this executioner caspase. The immunoblot analysis demonstrating the absence of caspase cleavage during cell death further confirmed these findings. OXY was also observed to induce the production of reactive oxygen species, which caused the depolarization of the mitochondrial membrane resulting in translocation of Apoptosis Inducing Factor (AIF) into the nucleus. Pretreatment of the cells with N-Acetyl Cysteine antioxidant prevented cell death resulting from OXY treatment. Thus, OXY initiates ROS-mediated, apoptosis-like cell death, involving mitochondrial membrane depolarization, translocation of AIF into the nucleus, and DNA fragmentation, resulting in caspase-independent cell death in MDA-MB-231 cells. The cytotoxicity manifested by OXY was also observed in 3D cell culture models and primary cells, thereby providing a basis for the utilization of OXY as a novel template for the future design of anticancer therapeutics.

Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy.

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target's binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP). We found that such new agents can effectively and rapidly form a covalent adduct with XIAP-BIR3 in vitro and in cell, approaching the rate of reaction, cellular permeability, and stability that are similar to what attained by acrylamides when targeting Cys residues. Our studies further validate aryl-fluorosulfates as valuable Lys-targeting electrophiles, for the design of inhibitors of both enzymes and protein-protein interactions.

Covalent Inhibitors of Protein-Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues.

We have recently reported a series of Lys-covalent agents targeting the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) using a benzamide-sulfonyl fluoride warhead. Using XIAP as a model system, we further investigated a variety of additional warheads that can be easily incorporated into binding peptides and analyzed their ability to form covalent adducts with lysine and other amino acids, including tyrosine, histidine, serine, and threonine, using biochemical and biophysical assays. Moreover, we tested aqueous, plasma stability, cell permeability, and cellular efficacy of the most effective agents. These studies identified aryl-fluoro sulfates as likely the most suitable electrophiles to effectively form covalent adducts with Lys, Tyr, and His residues, given that these agents were cell permeable and stable in aqueous buffer and in plasma. Our studies contain a number of general findings that open new possible avenues for the design of potent covalent protein-protein interaction antagonists.

Recent insights into natural product inhibitors of matrix metalloproteinases.

Members of the matrix metalloproteinase (MMP) family have biological functions that are central to human health and disease, and MMP inhibitors have been investigated for the treatment of cardiovascular disease, cancer and neurodegenerative disorders. The outcomes of initial clinical trials with the first generation of MMP inhibitors proved disappointing. However, our growing understanding of the complexities of the MMP function in disease, and an increased understanding of MMP protein architecture and control of activity now provide new opportunities and avenues to develop MMP-focused therapies. Natural products that affect MMP activities have been of strong interest as templates for drug discovery, and for their use as chemical tools to help delineate the roles of MMPs that still remain to be defined. Herein, we highlight the most recent discoveries of structurally diverse natural product inhibitors to these proteases.

A Heck-Based Strategy To Generate Anacardic Acids and Related Phenolic Lipids for Isoform-Specific Bioactivity Profiling.

A synthetic strategy for phenolic lipids such as anacardic acid and ginkgolic acid derivatives using an efficient and selective redox-relay Heck reaction followed by a stereoselective olefination is reported. This approach controls both the alkene position and stereochemistry, allowing the synthesis of natural and unnatural unsaturated lipids as single isomers. By this strategy, the activities of different anacardic acid and ginkgolic acid derivatives have been examined in a matrix metalloproteinase inhibition assay.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

An AAA+ ATPase Clamshell Targets Transposition.

DNA transposition plays key roles in genome diversity, pathogenesis, and evolution. Yet, structural and mechanistic information on transposition targeting and regulation is limited. Arias-Palomo and Berger now define the decameric organization of the AAA+ ATPase IstB, unveiling key insights into its targeting and regulation of IstA transposase activity.

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli.

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.

Human C6orf211 encodes Armt1, a protein carboxyl methyltransferase that targets PCNA and is linked to the DNA damage response.

Recent evidence supports the presence of an L-glutamyl methyltransferase(s) in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as "acidic residue methyltransferase-1" (Armt1), an enzyme that specifically targets proliferating cell nuclear antigen (PCNA) in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR).

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Developing advanced X-ray scattering methods combined with crystallography and computation.

The extensive use of small angle X-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one's repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions.