Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Tralokinumab Effectively Disrupts the IL-13/IL-13Rα1/IL-4Rα Signaling Complex but Not the IL-13/IL-13Rα2 Complex.

Tralokinumab, a fully human mAb specifically targeting the IL-13 cytokine, has demonstrated clinical efficacy and safety in patients with moderate-to-severe atopic dermatitis. Tralokinumab binds IL-13 with high affinity, which prevents the interaction of IL-13 with IL-13Rα1 and subsequent signaling. Similarly, tralokinumab-bound IL-13 cannot bind to IL-13Rα2, a proposed decoy receptor that is reported to bind IL-13 with extraordinarily high affinity. It has however not been fully elucidated to what extent tralokinumab interferes with the endogenous regulation of IL-13 through IL-13Rα2. In this mechanistic study, we used biophysical, biochemical, and cellular assays to investigate the effect of tralokinumab on the interaction between IL-13 and IL-13Rα1 and IL-13Rα2, respectively, as well as the effects on IL-13Rα2-mediated IL-13 internalization. We demonstrate that IL-13Rα2 binds IL-13 with exceptionally high affinity and that tralokinumab is unable to displace IL-13 from IL-13Rα2. In contrast to this, tralokinumab is able to disrupt the IL-13/IL-13Rα1 and IL-13Rα1/IL-13/IL-4Rα complex. Furthermore, we demonstrate that whereas the IL-13/tralokinumab complex is unable to bind IL-13Rα2, any IL-13 that is not bound by tralokinumab (i.e., free IL-13) can be bound by IL-13Rα2 and subsequently internalized, regardless of the presence of tralokinumab. In summary, our study indicates that tralokinumab does not interfere with endogenous IL-13Rα2-mediated regulation of free IL-13.

Synthesis of a Complex and Highly Potent PCSK9 Inhibitor.

The solution-based gram-scale synthesis of complex and highly potent proprotein convertase subtilisin-like/kexin type 9 (PCSK9) inhibitor 1 is presented. Construction of Northern fragment 2, followed by stepwise installation of Eastern 3, Southern 4, and Western 5 fragments, provided macrocyclic precursor 19. This intermediate was cross-linked via an intramolecular azide-alkyne click reaction, which preceded macrolactamization to afford the core framework of compound 1. Finally, coupling with poly(ethylene glycol) side-chain-based 6 gave the PCSK9 inhibitor 1.

Deep Anterior Lamellar Keratoplasty Over Penetrating Keratoplasty to Mitigate Positive Vitreous Pressure Complications.

This report describes deep anterior lamellar keratoplasty over penetrating keratoplasty (DALK-over-PKP) as an alternative technique to mitigate complications related to positive vitreous pressure (PVP) during PKP. We accomplished this by repairing the punctured cornea and performing a modified DALK where a full-thickness donor graft is placed over the host Descemet membrane, which is then removed after partial suturing of the graft. This mitigates the driving force behind the PVP by maintaining a closed-anterior chamber.

Total Synthesis of Darobactin A.

The collaborative total synthesis of darobactin A, a recently isolated antibiotic that selectively targets Gram-negative bacteria, has been accomplished in a convergent fashion with a longest linear sequence of 16 steps from d-Garner's aldehyde and l-serine. Scalable routes toward three non-canonical amino acids were developed to enable the synthesis. The closure of the bismacrocycle was realized through sequential, halogen-selective Larock indole syntheses, where the proper order of cyclizations proved crucial for the formation of the desired atropisomer of the natural product.

Metal-Free Stereoselective Synthesis of (E)- and (Z)-N-Monosubstituted ß-Aminoacrylates via Condensation Reactions of Carbamates.

N-monosubstituted ß-aminoacrylates are building blocks, which have been used in the preparation of amino acids and pharmaceuticals. Two efficient, stereoselective methods of preparation, via acid- or base-promoted condensation reactions of carbamates, are described. The base-promoted reaction is E-selective, while acid catalysis can, through the choice of solvent, selectively form E or Z. The acid-catalyzed E-selective process proceeds through a crystallization obviating the need for chromatographic purification.

Evaluation of 5-(Trifluoromethyl)-1,2,4-oxadiazole-Based Class IIa HDAC Inhibitors for Huntington's Disease.

Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12 acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound 12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of Huntington's disease.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Monthly Versus Treat-and-Extend Ranibizumab for Diabetic Macular Edema: A Prospective, Randomized Trial.

BACKGROUND AND OBJECTIVE: Compare fixed monthly dosing of ranibizumab to treat-and-extend (T&E) ranibizumab during a period of 24 months for diabetic macular edema (DME) treatment. PATIENTS AND METHODS: Single-center, randomized, prospective pilot study that included 20 eyes of 20 subjects. Patients' best-corrected visual acuity (BCVA) was less than or equal to 20/40 and central foveal thickness on spectral-domain optical coherence tomography was greater than 325 µm. Intravitreal ranibizumab was dosed monthly or by protocol-specified treat-and-extend. Primary outcome was mean change in mean BCVA. Institutional review board approval was obtained. RESULTS: At month 24 (M24), there was a mean 8.3-letter gain in the monthly treatment group and an 8.5-letter gain in the T&E group (P = .082; 90% confidence interval). The average change from baseline BCVA was not statistically significantly different at any timepoint. At M24, the median number of injections in the monthly and T&E groups were 22.5 and 18.5, respectively (P = .287). CONCLUSIONS: Visual acuity with monthly dosing appears equivalent to T&E dosing during the course of 24 months. There was a trend toward a lower injection burden in the T&E arm. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e191-e197.].

Thermal Shift as an Entropy-Driven Effect.

Thermal shift assays (TSAs) are among the most commonly used biophysical approaches in drug discovery in both academic and industrial settings. However, the most common interpretation of the data generated by a TSA is purely qualitative, using only the change in melting temperature (ΔTm) as a metric. This has left many questions surrounding the interpretation of the data as well as whether the TSA truly correlates with other assays. TSAs also lack theoretical descriptions of the melt behavior of proteins in the presence of multiple ligands. Here we describe a novel simplified analytical framework based on "pseudoisothermal" models as well as exact thermodynamic descriptions of protein-ligand melt behavior rooted in changes in the entropy of melting. We show how the models are broad and independently applicable, in that they can describe the behavior of any macromolecule such as a protein or DNA and demonstrate good correlations with other techniques. These models are shown to give good descriptions of assay systems containing single or multiple ligands and can determine the mechanism of interaction. The models are derived from first principles, and the theoretical justification is discussed.

Chemoselective Reduction of α-Cyano Carbonyl Compounds: Application to the Preparation of Heterocycles.

ß-Aminoacrylates are reactive intermediates that are useful building blocks in synthesis. General methods for their preparation typically afford α and ß disubstitution patterns or ß only. Molecules with only α-substituents (ß-hydrogen) are much less well-known. A chemoselective reductive tautomerization of α-cyanoacetates, using DIBAL-H, has been developed to access these valuable synthons. α,ß-Unsaturated cyanoacetates and α-cyanoketones can, also, be selectively reduced via this methodology. A series of heterocycles were prepared using these ß-enamino carbonyl compounds.

Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors.

Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.

Site selective syntheses of [(3)H]omeprazole using hydrogen isotope exchange chemistry.

Omeprazole (Prilosec®) is a selective and irreversible proton pump inhibitor used to treat various medical conditions related to the production of excess stomach acids. It functions by suppressing secretion of those acids. Radiolabeled compounds are commonly employed in the drug discovery and development process to support efforts including library screening, target identification, receptor binding, assay development and validation and safety assessment. Herein, we describe synthetic approaches to the controlled and selective labeling of omeprazole with tritium via hydrogen isotope exchange chemistry. The chemistry may also be used to prepare tritium labeled esomeprazole (Nexium®), the active pure (S)-enantiomer of omeprazole.

NMR-based approach to the analysis of radiopharmaceuticals: radiochemical purity, specific activity, and radioactive concentration values by proton and tritium NMR spectroscopy.

Compounds containing tritium are widely used across the drug discovery and development landscape. These materials are widely utilized because they can be efficiently synthesized and produced at high specific activity. Results from internally calibrated (3)H and (1)H nuclear magnetic resonance (NMR) spectroscopy suggests that at least in some cases, this calibrated approach could supplement or potentially replace radio-high-performance liquid chromatography for radiochemical purity, dilution and scintillation counting for the measurement of radioactivity per volume, and liquid chromatography/mass spectrometry analysis for the determination of specific activity. In summary, the NMR-derived values agreed with those from the standard approaches to within 1% to 9% for solution count and specific activity. Additionally, the NMR-derived values for radiochemical purity deviated by less than 5%. A benefit of this method is that these values may be calculated at the same time that (3)H NMR analysis provides the location and distribution of tritium atoms within the molecule. Presented and discussed here is the application of this method, advantages and disadvantages of the approach, and a rationale for utilizing internally calibrated (1)H and (3)H NMR spectroscopy for specific activity, radioactive concentration, and radiochemical purity whenever acquiring (3)H NMR for tritium location.

Recruiting U.S. and Canadian college students via social media for participation in a web-based brief intervention study.

OBJECTIVE: Recruiting young adults for health research is challenging. Social media provides wide access to potential research participants. We evaluated the feasibility of recruiting students via free message postings on Facebook and Twitter to participate in a web-based brief intervention study. The sample comprised students attending U.S. and Canadian universities. METHOD: During three semesters, institutional review board-approved recruitment messages were posted in 281 Facebook groups, 7 Facebook pages, and 27 message "tweets" on Twitter. RESULTS: A total of 708 eligible participants were recruited from Facebook. The mean enrollment rate per Facebook group was 0.21%; the rate was higher for host university groups (1.56%) compared with groups at other universities (0.10%). We recruited seven participants from Twitter. The sample was predominantly female (70%) with a mean age of 20.0 years. There were no significant differences between host university participants recruited through social media and traditional methods. The web-based intervention completion rate was 65%, and participants from the host university were more likely to complete the intervention than were groups at other universities (p = .01). CONCLUSIONS: Social media provides access to a large number of potential participants, and social media recruitment may be useful to researchers who can harness this broad reach. Facebook recruitment was feasible and free and resulted in a large number of enrolled participants. Social media recruitment for researchers at their own universities may be particularly fruitful. Despite wide access to students with Twitter, recruitment was slow. Social media recruitment allowed us to extend web-based intervention access to students in the United States and Canada.

Development of an automated system for preparation of liquid scintillation counting samples for radiolabeled pharmaceuticals.

Radiolabeled compounds are essential tools in drug development used to obtain critical metabolism and safety information. To support the synthesis and ensure quality of radiolabeled compounds for all programs, bench automation has been implemented in our laboratories. The concept of a platform technology for bench-top automation is not new. A considerable investment in the automation of various critical analytical laboratory workflows to both harmonize the efforts of a large and diverse global organization and minimize capital footprint has been made on our part. Various custom automation techniques and applications have been developed to increase capabilities and productivity of radiochemical analyses at Merck. In this paper, we will present a novel system that is capable of automating the liquid scintillation counting procedure. The system has handled multiple radiolabeled ((3)H, (14)C, and (35)S) pharmaceutical compounds with an accuracy of 5% with a standard deviation of 2% and a cycle time of ~10 min per analysis.

Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3.

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights.

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Mesalamine induced symptom exacerbation of ulcerative colitis: Case report and brief discussion.

This paper describes a rare case in which the oral administration of mesalamine resulted in the exacerbation of ulcerative colitis (UC) in a patient who was previously responsive to mesalamine and whose colitis had been in remission for eight years. Mesalamine and other 5-aminosalicylic acid compounds are the mainstay of treatment for UC; however up to 8% of patients are unable to take the medications due to intolerance or hypersensitivity reactions. Common drug reactions are fever, nausea, diarrhea and abdominal pain; however, exacerbation of UC has rarely been reported. This study highlights the importance of ruling out mesalamine as the causative agent in cases of UC exacerbations.

Universality in three- and four-body bound states of ultracold atoms.

Under certain circumstances, three or more interacting particles may form bound states. Although the general few-body problem is not analytically solvable, the so-called Efimov trimers appear for a system of three particles with resonant two-body interactions. The binding energies of these trimers are predicted to be universally connected to each other, independent of the microscopic details of the interaction. By exploiting a Feshbach resonance to widely tune the interactions between trapped ultracold lithium atoms, we find evidence for two universally connected Efimov trimers and their associated four-body bound states. A total of 11 precisely determined three- and four-body features are found in the inelastic-loss spectrum. Their relative locations on either side of the resonance agree well with universal theory, whereas a systematic deviation from universality is found when comparing features across the resonance.