A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Pulmonary translocation of ultrafine carbon particles in COPD and IPF patients.

OBJECTIVE: Epidemiological studies indicate association between elevated air pollution and adverse health effects. Several mechanisms have been suggested, including translocation of inhaled ultrafine carbon (UFC) particles into the bloodstream. Previous studies in healthy subjects have shown no significant pulmonary translocation of UFC-particles. This study aimed to assess if UFC-particles translocate from damaged alveolar compartment in subjects suffering from chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). METHODS: Eleven COPD and nine IPF subjects were exposed to a 100 nm UFC-particle-aerosol labeled with Indium-111. Activity in the body was followed up for 10 days using gamma camera planar-imaging as well as in blood and urine samples. RESULTS: The pulmonary central to periphery activity ratio was significantly higher for COPD as compared to IPF subjects at exposure, 1.8 and 1.4, respectively and remained constant throughout the test period. Ten days after exposure, the estimated median pulmonary translocation of UFC particles was 22.8 and 25.8% for COPD and IPF, respectively. Bound activity was present in blood throughout the test period, peaking at 24-h postinhalation with a median concentration of 5.6 and 8.9 Bq/ml for the COPD and IPF, respectively. Median bound activity excreted in urine (% of inhaled) after 10 days was 1.4% in COPD and 0.7% in IPF. Activity accumulation in liver and spleen could not be demonstrated. CONCLUSIONS: Our results suggest that UFC particles leak through the damaged alveolar barrier to the bloodstream in COPD and IPF patients probably distributing in a wide spectrum of whole-body tissues.

Discovery of novel N-1 substituted pyrazolopyrimidinones as potent, selective PDE2 inhibitors.

A focused SAR study was conducted on a series of N1-substituted pyrazolopyrimidinone PDE2 inhibitors to reveal compounds with excellent potency and selectivity. The series was derived from previously identified internal leads and designed to enhance steric interactions with key amino acids in the PDE2 binding pocket. Compound 26 was identified as a lead compound with excellent PDE2 selectivity and good physicochemical properties.

Development of indazole mineralocorticoid receptor antagonists and investigation into their selective late-stage functionalization.

The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability.

18F FDG-PET/CT evaluation of histological response after neoadjuvant treatment in patients with cancer of the esophagus or gastroesophageal junction.

BACKGROUND: In most parts of the world, curatively intended treatment for esophageal cancer includes neoadjuvant therapy, either with chemoradiotherapy or chemotherapy alone, followed by esophagectomy. Currently 18F-FDG positron emission tomography/computed tomography (PET/CT) is used for preoperative disease staging, but is not well established in the evaluation of neoadjuvant treatment. PURPOSE: To evaluate changes in PET parameters in relation to the histological primary tumor response in the surgical specimen in patients randomized to neoadjuvant chemoradiotherapy or chemotherapy. MATERIAL AND METHODS: Patients were randomized between either neoadjuvant chemotherapy or chemoradiotherapy followed by esophagectomy.18F-FDG PET/CT exams were conducted at baseline and following neoadjuvant treatment. Standardized uptake ratio (SUR) values were measured in the primary tumor and compared as regards histological responders and non-responders as well as different treatment arms. RESULTS: Seventy-nine patients were enrolled and 51 were available for analysis. A significant rate of SUR reduction was observed ( P = 0.02) in the primary tumor in histological responders compared to non-responders. Changes in SUR were significantly greater in responders following chemoradiotherapy ( P = 0.02), but not following chemotherapy alone ( P = 0.49). There was no statistically significant difference in SUR in patients with a complete histological response compared to those with a subtotal response. CONCLUSION: Our results are similar to those of previous studies and show that changes in the rate of SUR can be used reliably to differentiate histological responders from non-responders after neoadjuvant treatment with either chemoradiotherapy or chemotherapy. Limitations of current PET technology are likely to restrict the possibility of accurately ruling out limited residual disease.

Workflows and performances in the ranking prediction of 2016 D3R Grand Challenge 2: lessons learned from a collaborative effort.

The 2016 D3R Grand Challenge 2 includes both pose and affinity or ranking predictions. This article is focused exclusively on affinity predictions submitted to the D3R challenge from a collaborative effort of the modeling and informatics group. Our submissions include ranking of 102 ligands covering 4 different chemotypes against the FXR ligand binding domain structure, and the relative binding affinity predictions of the two designated free energy subsets of 15 and 18 compounds. Using all the complex structures prepared in the same way allowed us to cover many types of workflows and compare their performances effectively. We evaluated typical workflows used in our daily structure-based design modeling support, which include docking scores, force field-based scores, QM/MM, MMGBSA, MD-MMGBSA, and MacroModel interaction energy estimations. The best performing methods for the two free energy subsets are discussed. Our results suggest that affinity ranking still remains very challenging; that the knowledge of more structural information does not necessarily yield more accurate predictions; and that visual inspection and human intervention are considerably important for ranking. Knowledge of the mode of action and protein flexibility along with visualization tools that depict polar and hydrophobic maps are very useful for visual inspection. QM/MM-based workflows were found to be powerful in affinity ranking and are encouraged to be applied more often. The standardized input and output enable systematic analysis and support methodology development and improvement for high level blinded predictions.

Performance of multiple docking and refinement methods in the pose prediction D3R prospective Grand Challenge 2016.

We describe the performance of multiple pose prediction methods for the D3R 2016 Grand Challenge. The pose prediction challenge includes 36 ligands, which represent 4 chemotypes and some miscellaneous structures against the FXR ligand binding domain. In this study we use a mix of fully automated methods as well as human-guided methods with considerations of both the challenge data and publicly available data. The methods include ensemble docking, colony entropy pose prediction, target selection by molecular similarity, molecular dynamics guided pose refinement, and pose selection by visual inspection. We evaluated the success of our predictions by method, chemotype, and relevance of publicly available data. For the overall data set, ensemble docking, visual inspection, and molecular dynamics guided pose prediction performed the best with overall mean RMSDs of 2.4, 2.2, and 2.2 Å respectively. For several individual challenge molecules, the best performing method is evaluated in light of that particular ligand. We also describe the protein, ligand, and public information data preparations that are typical of our binding mode prediction workflow.

Predictive value of [18F]-fluoride PET for monitoring bone remodeling in patients with orthopedic conditions treated with a Taylor spatial frame.

PURPOSE: The Taylor Spatial Frame (TSF) is used to correct orthopedic conditions such as correction osteotomies in delayed fracture healing and pseudarthrosis. Long-term TSF-treatments are common and may lead to complications. Current conventional radiological methods are often unsatisfactory for therapy monitoring. Hence, an imaging technique capable of quantifying bone healing progression would be advantageous. METHODS: A cohort of 24 patients with different orthopedic conditions, pseudarthrosis (n = 10), deformities subjected to correction osteotomy (n = 9), and fracture (n = 5) underwent dynamic [18F]-fluoride (Na18F) PET/CT at 8 weeks and 4 months, respectively, after application of a TSF. Parametric images, corresponding to the net transport rate of [18F]-fluoride from plasma to bone, K i were calculated. The ratio of the maximum K i at PET scan 2 and 1 ([Formula: see text]) as well as the ratio of the maximum Standard Uptake Value at PET scan 2 and 1 ([Formula: see text]) were calculated for each individual. Different treatment end-points were scored, and the overall treatment outcome score was compared with the osteoblastic activity progression as scored with [Formula: see text] or [Formula: see text]. RESULTS: [Formula: see text] and [Formula: see text] were not correlated within each orthopedic group (p > 0.1 for all groups), nor for the pooled population (p = 0.12). The distribution of [Formula: see text] was found significantly different among the different orthopedic groups (p = 0.0046) -also for [Formula: see text] (p = 0.022). The positive and negative treatment predictive values for [Formula: see text] were 66.7 % and 77.8 %, respectively. Corresponding values for [Formula: see text] were 25 % and 33.3 % CONCLUSIONS: The [Formula: see text] obtained from dynamic [18F]-fluoride-PET imaging is a promising predictive factor to evaluate changes in bone healing in response to TSF treatment.

Discovery of novel non-steroidal reverse indole mineralocorticoid receptor antagonists.

Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.

Discovery of benzofuran propanoic acid GPR120 agonists: From uHTS hit to mechanism-based pharmacodynamic effects.

The transformation of an aryloxybutanoic acid ultra high-throughput screening (uHTS) hit into a potent and selective series of G-protein coupled receptor 120 (GPR120) agonists is reported. uHTS hit 1 demonstrated an excellent rodent pharmacokinetic profile and selectivity over the related fatty acid receptor GPR40, but only modest GPR120 potency. Optimization of the "left-hand" aryl group led to compound 6, which demonstrated a GPR120 mechanism-based pharmacodynamic effect in a mouse oral glucose tolerance test (oGTT). Further optimization gave rise to the benzofuran propanoic acid series (exemplified by compound 37), which demonstrated acute mechanism-based pharmacodynamic effects. The combination of in vivo efficacy and attractive rodent pharmacodynamic profiles suggests compounds generated from this series may afford attractive candidates for the treatment of Type 2 diabetes.

Heterocyclic core analogs of a direct thrombin inhibitor.

Thrombin is a serine protease that plays a key role in blood clotting. Pyrrolidine 1 is a potent thrombin inhibitor discovered at Merck several years ago. Seven analogs (2-8) of 1 in which the pyrrolidine core was replaced with various heterocycles were prepared and evaluated for activity against thrombin, clotting factors VIIa, IXa, Xa, and XIIa, and trypsin. The thiomorpholine analog 6 was the most active, essentially matching the thrombin inhibitory activity of 1 with slightly improved selectivity over trypsin.

Mineralocorticoid receptor antagonists: identification of heterocyclic amide replacements in the oxazolidinedione series.

Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure-activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists.

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.

First week of life respiratory management and pulmonary ventilation/perfusion matching in infants with bronchopulmonary dysplasia: a retrospective observational study.

OBJECTIVE: To investigate the association between early neonatal respiratory management in infants with bronchopulmonary dysplasia (BPD) and the degree of pulmonary ventilation perfusion-matching (V/Q) at term. METHODS: 30 preterm infants with a diagnosis of BPD who were initially treated with either controlled mechanical ventilation/continuous positive airway pressure (CMV/CPAP) (n = 14) or high-frequency oscillatory ventilation (HFOV) using a high lung-volume strategy (n = 16) were retrospectively included in this study. All infants underwent pulmonary V/Q single photon emission computed tomography at a median postmenstrual age of 37 weeks. RESULTS: Infants treated with HFOV had significantly larger proportion of the lung with matched V/Q as compared to infants treated with CMV/CPAP, median (interquartile range) 60.4% (55.5-66.0%) and 45.8% (37.8-53.1%) respectively (p = 0.01). CONCLUSIONS: In infants who needed mechanical ventilation the first week of life and later developed BPD an association was observed between treatment with a HFOV and better pulmonary V/Q matching at near-term age.

SUMO: In Silico Sequence Assessment Using Multiple Optimization Parameters.

To select the most promising screening hits from antibody and VHH display campaigns for subsequent in-depth profiling and optimization, it is highly desirable to assess and select sequences on properties beyond only their binding signals from the sorting process. In addition, developability risk criteria, sequence diversity, and the anticipated complexity for sequence optimization are relevant attributes for hit selection and optimization. Here, we describe an approach for the in silico developability assessment of antibody and VHH sequences. This method not only allows for ranking and filtering multiple sequences with regard to their predicted developability properties and diversity, but also visualizes relevant sequence and structural features of potentially problematic regions and thereby provides rationales and starting points for multi-parameter sequence optimization.

Selective Wee1 Inhibitors Led to Antitumor Activity In Vitro and Correlated with Myelosuppression.

Wee1 is a tyrosine kinase that is highly expressed in several cancer types. Wee1 inhibition can lead to suppression of tumor cell proliferation and sensitization of cells to the effects of DNA-damaging agents. AZD1775 is a nonselective Wee1 inhibitor for which myelosuppression has been observed as a dose-limiting toxicity. We have applied structure-based drug design (SBDD) to rapidly generate highly selective Wee1 inhibitors that demonstrate better selectivity than AZD1775 against PLK1, which is known to cause myelosuppression (including thrombocytopenia) when inhibited. While selective Wee1 inhibitors described herein still achieved in vitro antitumor efficacy, thrombocytopenia was still observed in vitro.

The pulmonary deposition and retention of indium-111 labeled ultrafine carbon particles in healthy individuals.

CONTEXT: Particulate air pollution, for example, from ultrafine (UF) particles, has negative health effects. However, there is still limited knowledge regarding the fate of inhaled particles in the human body. OBJECTIVES: To describe the normal lung deposition and 1 week particle retention of indium-111 labeled UF carbon particles in healthy subjects. Additionally, the possibility to extend the follow-up period to 4 weeks was also investigated for one of the subjects. RESULTS: The cumulative pulmonary particle clearance 1 week post-administration, corrected for activity leaching and mucocilliary transport of activity deposited in the central airways, was 4.3 ± 8.5% (average ± standard deviation at group level), with marginal translocation of particles from lungs to blood, 0.3%. There was no observable elimination of particles from the body via urine. Seven days after exposure, the cumulated activity leaching was 3% (group level), which indicates a stable bonding between the particles and Indium-111. The volunteer followed for a total of 4 weeks, showed a cumulative decrease of activity retention in the lungs of 10.5%. After correction for activity leaching and clearance from central airway deposition, the estimated particle clearance was about 2%. CONCLUSIONS: No evidence for particle translocation from the lungs could be proven 7 days after exposure. It is possible to follow-up Indium-111 labeled UF carbon particles at least 1 month post-administration without increasing the administered activity.

Primary hyperparathyroidism, a machine learning approach to identify multiglandular disease in patients with a single adenoma found at preoperative Sestamibi-SPECT/CT.

Objective: Successful preoperative image localisation of all parathyroid adenomas (PTA) in patients with primary hyperparathyroidism (pHPT) and multiglandular disease (MGD) remains challenging. We investigate whether a machine learning classifier (MLC) could predict the presence of overlooked PTA at preoperative localisation with 99mTc-Sestamibi-SPECT/CT in MGD patients. Design: This study is a retrospective study from a single tertiary referral hospital initially including 349 patients with biochemically confirmed pHPT and cured after surgical parathyroidectomy. Methods: A classification ensemble of decision trees with Bayesian hyperparameter optimisation and five-fold cross-validation was trained with six predictor variables: the preoperative plasma concentrations of parathyroid hormone, total calcium and thyroid-stimulating hormone, the serum concentration of ionised calcium, the 24-h urine calcium and the histopathological weight of the localised PTA at imaging. Two response classes were defined: patients with single-gland disease (SGD) correctly localised at imaging and MGD patients in whom only one PTA was localised on imaging. The data set was split into 70% for training and 30% for testing. The MLC was also tested on a subset of the original data based on CT image-derived PTA weights. Results: The MLC achieved an overall accuracy at validation of 90% with an area under the cross-validation receiver operating characteristic curve of 0.9. On test data, the MLC reached a 72% true-positive prediction rate for MGD patients and a misclassification rate of 6% for SGD patients. Similar results were obtained in the testing set with image-derived PTA weight. Conclusions: Artificial intelligence can aid in identifying patients with MGD for whom 99mTc-Sestamibi-SPECT/CT failed to visualise all PTAs.