The cellular and molecular basis of the spur development in Impatiens uliginosa.

The nectar spur is an important feature of pollination and ecological adaptation in flowering plants, and it is a key innovation to promote species diversity in certain plant lineages. The development mechanism of spurs varies among different plant taxa. As one of the largest angiosperm genera, we have little understanding of the mechanism of spur development in Impatiens. Here, we investigated the initiation and growth process of spurs of Impatiens uliginosa based on histology and hormone levels, and the roles of AUXIN BINDING PROTEIN (ABP) and extensin (EXT) in spur development were explored. Our results indicate that the spur development of I. uliginosa is composed of cell division and anisotropic cell elongation. Imbalances in spur proximal-distal cell division lead to the formation of curved structures. Endogenous hormones, such as auxin and cytokinins, were enriched at different developmental stages of spurs. IuABP knockdown led to an increase in spur curves and distortion of morphology. IuEXT knockdown resulted in reduced spur length and loss of curve and inner epidermal papillae structures. This study provides new insights into the mechanism of spur development in core eudicots.

Global-scale no-tillage impacts on soil aggregates and associated carbon and nitrogen concentrations in croplands: A meta-analysis.

No-tillage treatment, including no-tillage with straw retention (NTS) and without (NT), has been widely used as an efficient and sustainable alternative to conventional tillage with straw retention (CTS) and without (CT) and greatly affects soil physical quality and organic matter dynamics in cropland ecosystems. Although some studies have reported the effects of NTS on soil aggregate stability and soil organic carbon (SOC) concentration, the underlying mechanisms of how soil aggregates, aggregate-associated SOC and total nitrogen (TN) respond to no-tillage remain unclear. Through a global meta-analysis of 91 studies in cropland ecosystems, we evaluated the effects of no-tillage on soil aggregates and their associated SOC and TN concentrations. On average, no-tillage treatment significantly decreased the proportions of microaggregates (MA) by 21.4 % (95 % CI, -25.5 to -17.3 %) and silt+clay size particles (SIC) by 24.1 (95 % CI, -30.9 to -17.0 %), and increased the proportions of large macroaggregate (LA) by 49.5 % (95 % CI, 36.7-63.0 %) and small macroaggregate (SA) by 6.1 % (95 % CI, 2.0-10.9 %) compared to those in conventional tillage. The SOC concentrations for all three aggregate sizes increased significantly with no tillage: for LA by 28.2 % (95 % CI, 18.8-39.5 %), SA by 18.0 % (95 % CI, 12.8-23.3 %), and MA by 9.1 % (95 % CI, 2.6-16.8 %). TN also increased significantly for all sizes with no tillage, with LA by 13.6 % (95 % CI, 8.6-17.6 %), SA by 11.0 % (95 % CI, 5.0-17.0 %), MA by 11.7 % (95 % CI, 7.0-16.4 %), and SIC by 7.6 % (95 % CI, 2.4-13.8 %). The magnitude of the no-tillage treatment effect on soil aggregation, aggregate-associated SOC and TN varied with the environmental and experimental conditions. The positive effect on the proportions of LA occurred with initial soil organic matter (SOM) content >10 g kg-1, whereas SOM <10 g kg-1 did not change significantly. Additionally, the effect size of NTS compared with CTS was lower than that of NT compared with CT. These findings suggest that NTS may promote physically protective SOC accumulation through the formation of macroaggregates by reducing disturbance destruction and increasing plant-derived binding agents. The findings highlight that no-tillage may enhance the formation of soil aggregates and the associated SOC and TN concentrations in global cropland ecosystems.

[Effect of Deep Fertilization with Slow/Controlled Release Fertilizer on N Fate in Clayey Soil Wheat Field].

Clayey soil seriously affects water-holding capacity and nutrient movement. Adopting appropriate agronomic measures to optimize the distribution of soil inorganic nitrogen (SIN) and reduce the nitrogen (N) loss in this soil is the key to agricultural sustainable development. To clarify the effect of deep fertilization of slow/controlled release fertilizer with sowing on N loss in a clayey soil wheat field, two types of fertilizers, conventional fertilizer (CN) and slow/controlled release fertilizer (RCU), were selected in this study. Here, we evaluated the effects of these two fertilizer types on wheat yield, seasonal N runoff loss, ammonia volatilization, and N2O emissions in wheat fields in two typical fertilization modes (manual surface sowing and spreading (B) and belowground fertilization of slow/controlled release urea with mechanized strip sowing (D)). The temporal and spatial distribution characteristics of SIN in topsoil were also analyzed. The results showed that under the same fertilizer type, the wheat yield of D treatment was significantly higher than that of B treatment, whereas the yield of RCU was notably higher than that of CN under the same fertilization mode. D-RCU achieved the highest yield of 6.97 t·hm-2. The seasonal N losses from runoff and ammonia volatilization were higher than that from N2O emissions, and the responses of different N loss pathways to fertilizer types and fertilization methods were diverse. Fertilizer type and runoff occurrence time were the main influencing factors of N runoff loss, and N runoff loss of the RCU treatment was higher in the non-fertilization period. Unfortunately, affected by annual rainfall pattern, the seasonal N runoff loss of the RCU treatment (20.35 kg·hm-2) was significantly higher than that of the CN treatment (10.49 kg·hm-2). The late growth period was the main phase of ammonia volatilization, and the later period was jointly affected by fertilization modes and fertilizer types. The B-CN treatment induced the highest seasonal ammonia volatilization (18.15 kg·hm-2), which was significantly higher than that of the other treatments (7.31-8.38 kg·hm-2). Additionally, the D-RCU treatment (2.41 kg·hm-2) tended to reduce the N2O emissions in comparison to that in the B-CN treatment (4.02 kg·hm-2). The results also indicated that the horizontal movement of SIN was higher than the vertical movement. Deep fertilization of RCU was conducive to optimizing the spatial and temporal distribution of SIN, which was the main reason for the increase in wheat yield and the control of N loss from wheat fields. These results suggest that RCU is a suitable alternative fertilizer for increasing yield and reducing N loss in clayey soil wheat fields; D-RCU can increase the wheat yield and reduce ammonia volatilization and N2O emissions in wheat fields by optimizing the spatial and temporal distribution of SIN, and its increasing effect on N runoff loss in the non-fertilization period deserves attention.

Marker-assisted pyramiding of γ-tocopherol methyltransferase and glutamate formiminotransferase genes for development of biofortified sweet corn hybrids.

Micronutrients, including vitamins, minerals, and other bioactive compounds, have tremendous impacts on human health. Much progress has been made in improving the micronutrient content of inbred lines in various crops through biofortified breeding. However, biofortified breeding still falls short for the rapid generation of high-yielding hybrids rich in multiple micronutrients. Here, we bred multi-biofortified sweet corn hybrids efficiently through marker-assisted selection. Screening by molecular markers for vitamin E and folic acid, we obtained 15 inbred lines carrying favorable alleles (six for vitamin E, nine for folic acid, and three for both). Multiple biofortified corn hybrids were developed through crossing and genetic diversity analysis.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Augmenting Hit Identification by Virtual Screening Techniques in Small Molecule Drug Discovery.

Two orthogonal approaches for hit identification in drug discovery are large-scale in vitro and in silico screening. In recent years, due to the emergence of new targets and a rapid increase in the size of the readily synthesizable chemical space, there is a growing emphasis on the integration of the two techniques to improve the hit finding efficiency. Here, we highlight three examples of drug discovery projects at Merck & Co., Inc., Kenilworth, NJ, USA in which different virtual screening (VS) techniques, each specifically tailored to leverage knowledge available for the target, were utilized to augment the selection of high-quality chemical matter for in vitro assays and to enhance the diversity and tractability of hits. Central to success is a fully integrated workflow combining in silico and experimental expertise at every stage of the hit identification process. We advocate that workflows encompassing VS as part of an integrated hit finding plan should be widely adopted to accelerate hit identification and foster cross-functional collaborations in modern drug discovery.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice.

AIMS: Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity. METHODS: We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy. RESULTS: Firstly, using a combination of published crystal structures and in-house data, we demonstrated that the binding site utilized by all of the DPP-4 inhibitors assessed was the same as that used by neuropeptide Y, supporting the hypothesis that DPP-4 inhibitors are able to competitively inhibit endogenous substrates for the enzyme. Secondly, we ascertained that the enzymatic cleft of DPP-4 is a relatively large cavity which displays conformational flexibility to accommodate structurally diverse inhibitor molecules. Finally, we found that for all inhibitors, irrespective of their chemical structure, the inhibition of plasma DPP-4 enzyme activity correlates directly with acute plasma glucose lowering in mice. CONCLUSION: The common binding site utilized by different DPP-4 inhibitors enables similar competitive inhibition of the cleavage of the endogenous DPP-4 substrates. Furthermore, despite chemical diversity and a range of binding potencies observed amongst the DPP-4 inhibitors, a direct relationship between enzyme inhibition in the plasma and glucose lowering is evident in mice for each member of the classes studied.

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.

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis.

8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.

Discovery of novel BTK inhibitors with carboxylic acids.

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Discovery of Novel Tricyclic Heterocycles as Potent and Selective DPP-4 Inhibitors for the Treatment of Type 2 Diabetes.

In our efforts to develop second generation DPP-4 inhibitors, we endeavored to identify distinct structures with long-acting (once weekly) potential. Taking advantage of X-ray cocrystal structures of sitagliptin and other DPP-4 inhibitors, such as alogliptin and linagliptin bound to DPP-4, and aided by molecular modeling, we designed several series of heterocyclic compounds as initial targets. During their synthesis, an unexpected chemical transformation provided a novel tricyclic scaffold that was beyond our original design. Capitalizing on this serendipitous discovery, we have elaborated this scaffold into a very potent and selective DPP-4 inhibitor lead series, as highlighted by compound 17c.

The discovery of novel 5,6,5- and 5,5,6-tricyclic pyrrolidines as potent and selective DPP-4 inhibitors.

Novel potent and selective 5,6,5- and 5,5,6-tricyclic pyrrolidine dipeptidyl peptidase IV (DPP-4) inhibitors were identified. Structure-activity relationship (SAR) efforts focused on improving the intrinsic DPP-4 inhibition potency, increasing protease selectivity, and demonstrating clean ion channel and cytochrome P450 profiles while trying to achieve a pharmacokinetic profile suitable for once weekly dosing in humans.

Discovery of 8-Amino-imidazo[1,5-a]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.

[Temporal and Spatial Distribution of Phosphorus in Paddy Fields Under Cyclic Irrigation of Drainage Water].

Considering the potential of cyclic irrigation to increase rainfall use efficiency and reduce agricultural non-point pollution, the experiment of phosphorus transport in paddy fields under cyclic irrigation of drainage water was conducted to address the problem of phosphorus loss pollution and the mismatch between rainfall temporal distribution and crop requirement in the south of China. Lotus pond water and fishpond water were used to irrigate paddy fields for monitoring concentrations of total phosphorus (TP), dissolved phosphorus (DP), and dissolved reactive phosphorus (DRP) in surface water and leachate, and soil profile total phosphorus and Olsen-P concentrations. The results showed that the concentrations of TP, DP and DRP in surface water and leachate decreased along the field under cyclic irrigation of drainage water, especially the phosphorus concentrations of leachate dropped more obviously. As the phosphorus content of cyclic irrigation water sources varied within a certain range, phosphorus concentrations of surface water and leachate did not increased. The concentrations of TP, DP and DRP in surface water and leachate varied with cyclic irrigation time, and the least phosphorus concentrations were observed in August. Top soil Olsen-P concentration decreased along the field and increased with phosphorus content of cyclic irrigation water sources, and soil profile TP concentration was not influenced by cyclic irrigation. Phosphorus removal ratio of paddy field could be increased by extending field length or cyclic irrigation in August.

Structure-activity-relationship of amide and sulfonamide analogs of omarigliptin.

A series of novel substituted-[(3R)-amino-2-(2,5-difluorophenyl)]tetrahydro-2H-pyran analogs have been prepared and evaluated as potent, selective and orally active DPP-4 inhibitors. These efforts lead to the discovery of a long acting DPP-4 inhibitor, omarigliptin (MK-3102), which recently completed phase III clinical development and has been approved in Japan.

Omarigliptin (MK-3102): a novel long-acting DPP-4 inhibitor for once-weekly treatment of type 2 diabetes.

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2- (2,5-difluorophenyl)-5-(4,6-dihydropyrrolo [3,4-c]pyrazol-5-(1H)-yl)tetrahydro-2H-pyran-3-amine (23) [corrected].

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.

The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization.

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.

Discovery of benzodiazepine sulfonamide-based bombesin receptor subtype 3 agonists and their unusual chirality.

We report herein the discovery of benzodiazepine sulfonamide-based bombesin receptor subtype 3 (BRS-3) agonists and their unusual chirality. Starting from a high-throughput screening lead, we prepared a series of BRS-3 agonists with improved potency and pharmacokinetic properties, of which compound 8a caused mechanism-based, dose-dependent food intake reduction and body weight loss after oral dosing in diet-induced obese mice. This effort also led to the discovery of a novel family of chiral molecules originated from the conformationally constrained seven-membered diazepine ring.