Geographic Equity and Environmental Sustainability of Conference Models: Results of a Comparative Analysis.

We conducted a comparative analysis of in-person, virtual, and hybrid conferences on tuberculosis hosted by Keystone Symposia and examined the number of participants, their country of residence, carbon dioxide equivalents (CO2e) produced, and participant impressions regarding scientific quality. Data were available from three in-person meetings, one virtual meeting, and one hybrid. The virtual conference hosted 2.5-fold more participants compared with the in-person conferences (842 versus an average of 328) from more than double the number of countries (68 versus an average of 33). The virtual conference attracted 4.5-fold more participants from countries with a high burden of tuberculosis, compared with the average in-person conference (209 versus an average of 46). For in-person meetings, an average of 79% of participants were based in high-income countries. For the virtual meeting, 53% of participants were from high-income countries, and 47% from low- and middle-income countries. For the hybrid conference, there were 465 participants from 43 countries, of which 289 attended in person from a total of 20 countries, and 176 participated virtually from 34 countries. Of those who took part in person, 91% were from high-income countries. The average CO2e emissions from an in-person conference was 696 tons of CO2e, with 96.0% from air travel. The virtual meeting produced 0.48 ton of CO2e from electricity usage, a 1,450-fold decrease compared with in-person events. Virtual conferences scored a content quality rating of 87.3% to 90.8% compared with a range of 86.4% to 92.2% for in-person conferences.

Reimagining scientific conferences-a Keystone Symposia report.

Scientific conferences play an important role in advancing research, scholarship, and the careers of emerging scientists. The COVID-19 pandemic offered meeting organizers and researchers alike an opportunity to reimagine what scientific conferences could look like. Virtual conferences can increase inclusivity and accessibility while decreasing costs and carbon emissions. However, it is generally perceived that the digital world fails to adequately recapitulate many of the benefits of in-person face-to-face interactions; these include socializing, and collaborative environments that can forge new research directions and provide critical career development opportunities. On November 15 and 16, 2022, researchers, representatives from diverse scientific conference organizations, leaders in virtual platform technologies, and innovators in conference design gathered online for the Open Access Keystone eSymposium "Reimagining Scientific Conferences." The meeting focused on how conference organizers can leverage lessons from the pandemic and emerging virtual platforms to engage new audiences, rethink strategies for scientific exchange, and decrease the carbon footprint of in-person events.

Microbiological profile, preclinical pharmacokinetics and efficacy of CRS0393, a novel antimycobacterial agent targeting MmpL3.

The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 µg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.

Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota.

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC90 value of 1 µg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides, bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.).

Optimization and Lead Selection of Benzothiazole Amide Analogs Toward a Novel Antimycobacterial Agent.

Mycobacteria remain an important problem worldwide, especially drug resistant human pathogens. Novel therapeutics are urgently needed to tackle both drug-resistant tuberculosis (TB) and difficult-to-treat infections with nontuberculous mycobacteria (NTM). Benzothiazole adamantyl amide had previously emerged as a high throughput screening hit against M. tuberculosis (Mtb) and was subsequently found to be active against NTM as well. For lead optimization, we applied an iterative process of design, synthesis and screening of several 100 analogs to improve antibacterial potency as well as physicochemical and pharmacological properties to ultimately achieve efficacy. Replacement of the adamantyl group with cyclohexyl derivatives, including bicyclic moieties, resulted in advanced lead compounds that showed excellent potency and a mycobacteria-specific spectrum of activity. MIC values ranged from 0.03 to 0.12 µg/mL against M. abscessus (Mabs) and other rapid- growing NTM, 1-2 µg/mL against M. avium complex (MAC), and 0.12-0.5 µg/mL against Mtb. No pre-existing resistance was found in a collection of n = 54 clinical isolates of rapid-growing NTM. Unlike many antibacterial agents commonly used to treat mycobacterial infections, benzothiazole amides demonstrated bactericidal effects against both Mtb and Mabs. Metabolic labeling provided evidence that the compounds affect the transfer of mycolic acids to their cell envelope acceptors in mycobacteria. Mapping of resistance mutations pointed to the trehalose monomycolate transporter (MmpL3) as the most likely target. In vivo efficacy and tolerability of a benzothiazole amide was demonstrated in a mouse model of chronic NTM lung infection with Mabs. Once daily dosing over 4 weeks by intrapulmonary microspray administration as 5% corn oil/saline emulsion achieved statistically significant CFU reductions compared to vehicle control and non-inferiority compared to azithromycin. The benzothiazole amides hold promise for development of a novel therapeutic agent with broad antimycobacterial activity, though further work is needed to develop drug formulations for direct intrapulmonary delivery via aerosol.

Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor.

Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents.

Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the "diversity gap" between nucleic acid-based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics.

Chemically modified DNA aptamers bind interleukin-6 with high affinity and inhibit signaling by blocking its interaction with interleukin-6 receptor.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

Crystal structure of interleukin-6 in complex with a modified nucleic acid ligand.

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (Kd = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (Kd = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (Kd = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.

Highly multiplexed proteomic platform for biomarker discovery, diagnostics, and therapeutics.

Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to the discovery of new biomarkers for diagnostics, new targets for therapeutics, and can identify patients most likely to benefit from treatment. Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of "real-time" health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as both folded binding entities with defined shapes and unique nucleic acid sequences recognizable by specific hybridization probes. Currently, our assay is capable of simultaneously measuring 1,030 proteins, extending to sub-pM detection limits, an average dynamic range of each analyte in the assay of > 3 logs, an overall dynamic range of at least 7 logs, and a throughput of one million analytes per week. Our collection includes SOMAmers that specifically recognize most of the complement cascade proteins. We have used this assay to identify potential biomarkers in a range of diseases such as malignancies, cardiovascular disorders, and inflammatory conditions. In this chapter, we describe the application of our technology to discovering large-scale protein expression changes associated with chronic kidney disease and non-small cell lung cancer. With this new proteomics technology-which is fast, economical, highly scalable, and flexible--we now have a powerful tool that enables whole-proteome proteomics, biomarker discovery, and advancing the next generation of evidence-based, "personalized" diagnostics and therapeutics.

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets.

Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.

Protein signature of lung cancer tissues.

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.

Rapid histochemistry using slow off-rate modified aptamers with anionic competition.

Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

Discovery and analysis of 4H-pyridopyrimidines, a class of selective bacterial protein synthesis inhibitors.

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyl-tRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC(50)s) ranging from 3 to 14 µM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 µg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition.

Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections.

OBJECTIVES: The aim of this study was to characterize the antimicrobial profile of REP3123, a novel inhibitor of methionyl-tRNA synthetase (MetRS) in development for the treatment of Clostridium difficile infection. METHODS: The spectrum of activity of REP3123 was determined by susceptibility testing of C. difficile and non-target organisms. The mode of action was studied by enzyme inhibition assays, macromolecular synthesis assays, target overexpression and selection of spontaneous resistant mutants. RESULTS: REP3123 was active against a collection of 108 clinical isolates of C. difficile and against epidemic, moxifloxacin-resistant BI/NAP1/027 strains (MIC range=0.5-1 mg/L and MIC(90) = 1 mg/L). The spectrum of activity included clinically important aerobic Gram-positive cocci such as Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Enterococcus faecium (MIC(90)s < 1 mg/L), but REP3123 was not active against most Gram-negative bacteria. REP3123 targeted C. difficile MetRS with a calculated inhibition constant (K(i)) of 0.020 nM, and selectivity was >1000-fold over human mitochondrial and cytoplasmic MetRS. The specific mode of action within bacterial cells was demonstrated by macromolecular synthesis assays that showed inhibition of protein synthesis by REP3123, and by metS overexpression, which resulted in a 16-fold increase in MIC for REP3123. Spontaneous REP3123-resistant mutants of C. difficile (MICs, 4-128 mg/L) arose with frequencies of 10(-8)-10(-9) and harboured distinct point mutations within the metS gene, resulting in 13 different amino acid substitutions. Most of the MetRS substitutions caused reduced catalytic efficiency and a growth fitness burden. CONCLUSIONS: REP3123 demonstrated a favourable microbiological profile and was found to target C. difficile with high specificity and selectivity.

Inhibition of methionyl-tRNA synthetase by REP8839 and effects of resistance mutations on enzyme activity.

REP8839 is a selective inhibitor of methionyl-tRNA synthetase (MetRS) with antibacterial activity against a variety of gram-positive organisms. We determined REP8839 potency against Staphylococcus aureus MetRS and assessed its selectivity for bacterial versus human orthologs of MetRS. The inhibition constant (K(i)) of REP8839 was 10 pM for Staphylococcus aureus MetRS. Inhibition of MetRS by REP8839 was competitive with methionine and uncompetitive with ATP. Thus, high physiological ATP levels would actually facilitate optimal binding of the inhibitor. While many gram-positive bacteria, such as Staphylococcus aureus, express exclusively the MetRS1 subtype, many gram-negative bacteria express an alternative homolog called MetRS2. Some gram-positive bacteria, such as Streptococcus pneumoniae and Bacillus anthracis, express both MetRS1 and MetRS2. MetRS2 orthologs were considerably less susceptible to REP8839 inhibition. REP8839 inhibition of human mitochondrial MetRS was 1,000-fold weaker than inhibition of Staphylococcus aureus MetRS; inhibition of human cytoplasmic MetRS was not detectable, corresponding to >1,000,000-fold selectivity for the bacterial target relative to its cytoplasmic counterpart. Mutations in MetRS that confer reduced susceptibility to REP8839 were examined. The mutant MetRS enzymes generally exhibited substantially impaired catalytic activity, particularly in aminoacylation turnover rates. REP8839 K(i) values ranged from 4- to 190,000-fold higher for the mutant enzymes than for wild-type MetRS. These observations provide a potential mechanistic explanation for the reduced growth fitness observed with MetRS mutant strains relative to that with wild-type Staphylococcus aureus.

Structure of PolC reveals unique DNA binding and fidelity determinants.

PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-A resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique beta-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.

Discovery and characterization of the cryptic psi subunit of the pseudomonad DNA replicase.

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of alpha and epsilon (polymerase and editing nuclease), beta (processivity factor), and the essential tau, delta, and delta' components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, chi and Psi are tightly associated clamp loader accessory subunits. The addition of E. coli chiPsi to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of chi and Psi counterparts in P. aeruginosa. The P. aeruginosa chi subunit was recognizable from sequence similarity, but Psi was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Psi subunit, encoded by holD. P. aeruginosa chi and Psi were co-expressed and purified as a 1:1 complex. P. aeruginosa chiPsi increased the specific activity of tau(3)deltadelta' 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between chiPsi and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Psi allowed us to identify Psi subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Psi family and confirms the existence of Psi in several organisms in which Psi was not identifiable based on sequence similarity alone.

Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors.

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (beta), single-stranded DNA-binding protein, a complex containing the polymerase (alpha) and exonuclease (epsilon) subunits, and the essential components of the DnaX complex (tau(3)deltadelta'). Efficient primer elongation requires the presence of alphaepsilon, beta, and tau(3)deltadelta'. Pseudomonas aeruginosa alphaepsilon can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas beta and tau(3)deltadelta' exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli psi subunit was not apparent in sequence similarity searches, addition of purified E. coli chi and psi (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas tau(3)deltadelta' complex approximately 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.