Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration.

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.

Computational Site Saturation Mutagenesis of Canonical and Non-Canonical Amino Acids to Probe Protein-Peptide Interactions.

Technologies for discovering peptides as potential therapeutics have rapidly advanced in recent years with significant interest from both academic and pharmaceutical labs. These advancements in turn drive the need for new computational tools to design peptides for purposes of advancing lead molecules into the clinic. Here we report the development and application of a new automated tool, AutoRotLib, for parameterizing a diverse set of non-canonical amino acids (NCAAs), N-methyl, or peptoid residues for use with the computational design program Rosetta. In addition, we developed a protocol for designing thioether-cyclized macrocycles within Rosetta, due to their common application in mRNA display using the RaPID platform. To evaluate the utility of these new computational tools, we screened a library of canonical and NCAAs on both a linear peptide and a thioether macrocycle, allowing us to quickly identify mutations that affect peptide binding and subsequently measure our results against previously published data. We anticipate in silico screening of peptides against a diverse chemical space will be a fundamental component for peptide design and optimization, as more amino acids can be explored in a single in silico screen than an in vitro screen. As such, these tools will enable maturation of peptide affinity for protein targets of interest and optimization of peptide pharmacokinetics for therapeutic applications.

Incorporating NOE-Derived Distances in Conformer Generation of Cyclic Peptides with Distance Geometry.

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Acceptability of self-collecting oropharyngeal swabs for sexually transmissible infection testing among men and women.

In 2016, the rate of USA gonorrhoea and chlamydia cases increased by 18.6% and 6.9% respectively. Most people infected are asymptomatic and are not treated immediately, which negatively affects sexually transmissible infection (STI)/HIV rates. Men and women were asked to provide self-collected oropharyngeal specimens for STI testing (n = 79). Over 75% reported the collection of the swab was 'easy' or 'very easy' to use; 90% were willing to test for STIs at home in the future. Self-collecting oropharyngeal swabs for STI testing is acceptable among men and women. Future research should test the effect of self-collecting pharyngeal swabs on STI testing behaviours and results.

Targeting the Hippo Pathway and Cancer through the TEAD Family of Transcription Factors.

The Hippo pathway is a critical transcriptional signaling pathway that regulates cell growth, proliferation and organ development. The transcriptional enhanced associate domain (TEAD) protein family consists of four paralogous transcription factors that function to modulate gene expression in response to the Hippo signaling pathway. Transcriptional activation of these proteins occurs upon binding to the co-activator YAP/TAZ whose entry into the nucleus is regulated by Lats1/2 kinase. In recent years, it has become apparent that the dysregulation and/or overexpression of Hippo pathway effectors is implicated in a wide range of cancers, including prostate, gastric and liver cancer. A large body of work has been dedicated to understanding the therapeutic potential of modulating the phosphorylation and localization of YAP/TAZ. However, YAP/TAZ are considered to be natively unfolded and may be intractable as drug targets. Therefore, TEAD proteins present themselves as an excellent therapeutic target for intervention of the Hippo pathway. This review summarizes the functional role of TEAD proteins in cancer and assesses the therapeutic potential of antagonizing TEAD function in vivo.

An evaluation of the SD Bioline HIV/syphilis duo test.

Many health agencies now recommend routine HIV and syphilis testing for pregnant women and most-at-risk populations such as men who have sex with men. With the increased availability of highly sensitive, low cost rapid point-of-care tests, the ability to meet those recommendations has increased, granting wider access to quick and accurate diagnoses. Using blood specimens collected from a Baltimore City Health Department (BCHD) sexually transmitted infection clinic, we evaluated the SD Bioline HIV/Syphilis Duo, a rapid test that simultaneously detects antibodies to HIV and syphilis and has the potential to further benefit clinics and patients by reducing costs, testing complexity, and patient wait times. SD DUO HIV sensitivity and specificity, when compared to BCHD results, were 91.7 and 99.5%, respectively. SD DUO syphilis sensitivity and specificity, when compared to rapid plasma reagin, were 85.7 and 96.8%, respectively, and 69.7 and 99.7%, respectively, when compared to Treponema pallidum particle agglutination (TPPA). SD DUO syphilis sensitivity and specificity, when compared to a traditional screening algorithm, improved to 92.3 and 100%, respectively, and improved to 72.9 and 99.7%, respectively, when compared to a reverse screening algorithm. The HIV component of the SD DUO performed moderately well. However, results for the SD DUO syphilis component, when compared to TPPA, support the need for further testing and assessment.

Stability Studies on Dry Swabs and Wet Mailed Swabs for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Aptima Assays.

The Aptima Combo 2 (AC2) and Aptima CT (ACT) (Hologic Inc., San Diego, CA) are nucleic acid amplification tests (NAATs) that detect Chlamydia trachomatis AC2 also detects Neisseria gonorrhoeae Storage and temperature conditions may impact the utility of NAATs in some settings and screening programs. We evaluated specimen stability for use beyond the Aptima package insert specifications for temperature and duration of storage (between 2°C and 30°C and 60 days, respectively) in two studies: (i) dry C. trachomatis-seeded swabs were used with ACT after storage at 4°C, 23°C, or 36°C for up to 84 days and (ii) swabs seeded with C. trachomatis and N. gonorrhoeae and then placed in transport medium were tested with AC2, after being mailed via the U.S. Postal Service to three different sites. Prolonged storage of samples had no effect, and samples stored at 4°C, 23°C, and 36°C for up to 84 days yielded comparable ACT positivities, although there was a drop in signal intensity for virtually all specimens under all storage/shipping conditions after day 21. In the mailing study, 80%, 52% and 29% of seeded swabs were exposed to temperatures of >30°C during three rounds in transit, and 2% reached temperatures of >40°C. No evidence of signal degradation in the AC2 assay for detection of C. trachomatis or N. gonorrhoeae was observed, although some mailed swabs took more than 5 weeks to reach the laboratory site. These two studies support the potential use of swabs at temperatures above 36°C and storage beyond 60 days and provide confidence regarding this commercially available NAAT for testing of specimens after mailing.

Targeting Bacterial Nitric Oxide Synthase with Aminoquinoline-Based Inhibitors.

Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.

Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.

Structure-based design of bacterial nitric oxide synthase inhibitors.

Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

Identification of redox partners and development of a novel chimeric bacterial nitric oxide synthase for structure activity analyses.

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.

Structural and biological studies on bacterial nitric oxide synthase inhibitors.

Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

Evaluation of a novel electrochemical detection method for Chlamydia trachomatis: application for point-of-care diagnostics.

Atlas Genetics has developed a point-of-care device for Chlamydia trachomatis utilizing a novel electrochemical detection principle. The assay has a time-to-result of less than 25 min. An independent preclinical validation study using 306 pretyped clinical samples determined a clinical sensitivity of 98.1% and specificity of 98.0%.

Chlamydia pneumoniae infection and risk for lung cancer.

BACKGROUND: We evaluated the relationship of Chlamydia pneumoniae infection with prospective lung cancer risk using traditional serologic markers [microimmunoflourescence (MIF) IgG and IgA antibodies] and Chlamydia heat shock protein-60 (CHSP-60) antibodies, a marker for chronic chlamydial infection. METHODS: We conducted a nested case-control study (593 lung cancers and 671 controls) within the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (N = 77,464). Controls were matched to cases by age, sex, randomization year, follow-up time, and smoking (pack-years of smoking, time since quitting). We assessed C. pneumoniae seropositivity and endpoint antibody titers (IgG and IgA against C. pneumoniae elementary bodies and IgG against CHSP-60). RESULTS: C. pneumoniae seropositivity by microimmunoflourescence IgG or IgA antibodies was not associated with lung cancer [odds ratio of 0.88 and 95% confidence interval (95% CI) of 0.69-1.13 for IgG; odds ratio of 0.98 and 95% CI of 0.75-1.27 for IgA]. In contrast, individuals seropositive for CHSP-60 IgG antibodies had significantly increased lung cancer risk (odds ratio, 1.30; 95% CI, 1.02-1.67), and risk increased with increasing antibody titers (P trend = 0.006). CHSP-60-related risk did not differ significantly by lung cancer histology, follow-up time, or smoking. CHSP-60 seropositivity was associated with increased risk 2 to 5 years before lung cancer diagnosis (odds ratio, 1.77; 95% CI, 1.16-2.71; P trend = 0.006), thus arguing against reverse causality. CONCLUSIONS: CHSP-60 seropositivity and elevated antibody titers were associated with significantly increased risk for subsequent lung cancer, supporting an etiologic role for C. pneumoniae infection in lung carcinogenesis. IMPACT: Our results highlight the potential for lung cancer risk reduction through treatments targeted toward C. pneumoniae infections and chronic pulmonary inflammation.

Airway complication following functional endoscopic sinus surgery.

Difficulty breathing after upper airway surgery requires immediate evaluation and treatment. We present a case of airway compromise after sinus surgery due to edema of the uvula. The patient was admitted for observation overnight and discharged the next day. A discussion of potential airway changes after sinonasal surgery is presented.