Reversible male contraception by targeted inhibition of serine/threonine kinase 33.

Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.

Discovery of Highly Potent and BMPR2-Selective Kinase Inhibitors Using DNA-Encoded Chemical Library Screening.

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor ß (TGFß) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion "unbiased" and "kinase-biased" DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure-activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors.

Evaluation of Natural Pigments Production in Response to Various Stress Signals in Cell Lines of Stenocereus queretaroensis.

Stenocereus queretaroensis (F.A.C. Weber ex Mathes.) Buxb is a cactus that has long been used as a source food in central and northern México. Its fruits, commonly called pitayas, biosynthesize high amounts of betalains. These molecules are water-soluble nitrogenous compounds; that compared to other pigments, such as anthocyanins or carotenoids, stand out for their physicochemical stability in industrial processes. Due to genetic and environmental factors involved in the biosynthesis and accumulation of secondary metabolites in plants, we tested different stress-inducing agents (elicitor, osmotic, salt, and temperature) to induce betalains accumulation in cell culture from fruits of Stenocereus queretaroensis. This work aimed to understand stress conditions that induce the metabolic pathways required for the accumulation of betalains. The results show how betacyanin concentration increases under high sugar conditions, thus affecting the expression of L-DOPA 4, 5 dioxygenase resulting in a strong dark red coloration. This suggests this enzyme is part of a rate-limiting step in betalain production. In addition, we found that betalains accumulation occurs under particular stress conditions. Cells that have a high level of betacyanins show better resistance to stress in the cell culture, as well as an overall different behavior including cell aggregation and alterations in nuclear size. Together the results shown here may provide new strategies to manipulate and mass produce the pigments from Stenocereus queretaroensis in cell culture.

Discovery and characterization of bromodomain 2-specific inhibitors of BRDT.

Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.

Mycophenolic anilides as broad specificity inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.

C-N Coupling of DNA-Conjugated (Hetero)aryl Bromides and Chlorides for DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical library (DECL) screens are a rapid and economical tool to identify chemical starting points for drug discovery. As a robust transformation for drug discovery, palladium-catalyzed C-N coupling is a valuable synthetic method for the construction of DECL chemical matter; however, currently disclosed methods have only been demonstrated on DNA-attached (hetero)aromatic iodide and bromide electrophiles. We developed conditions utilizing an N-heterocyclic carbene-palladium catalyst that extends this reaction to the coupling of DNA-conjugated (hetero)aromatic chlorides with (hetero)aromatic and select aliphatic amine nucleophiles. In addition, we evaluated steric and electronic effects within this catalyst series, carried out a large substrate scope study on two representative (hetero)aryl bromides, and applied this newly developed method within the construction of a 63 million-membered DECL.

Quasi-static and dynamic response, and texture evolution of two overaged Al 7056 alloy plates in T761 and T721 tempers: Experiments and modeling.

The thermo-mechanical behavior and texture evolution of two overaged Al 7056 alloy plates, in T761 and T721 tempers, are measured over a wide range of strain rates (10‒4 - 3 × 103 s‒1) and temperatures (22-300 °C) under uniaxial tension and compression along the thickness direction, i.e. normal to the plate surface. A detailed study of the initial microstructure reveals an increase in precipitate size and decrease in density of precipitates, as the alloy is aged from the T761 to T721 temper; which in turn affects the flow stress and strain hardening behavior. Differences in flow strength and strain hardening rate, as well as tension-compression asymmetry in the two tempers, are apparent at the lower temperatures (22 °C & 100 °C) and decrease significantly at the higher temperatures (200 °C & 300 °C). Furthermore, initial texture measurements show a strong texture gradient along the normal direction (ND) of the plate. This texture gradient affects the ultimate stress insignificantly. However, it does have a considerable effect on the failure strains of specimens taken from different locations through the thickness. A transition from shear fracture at and below 200 °C to cup and cone fracture mode above 200 °C is observed in tension. Both tempers exhibit a positive strain rate sensitivity (SRS) that is dependent on temperature and strain rate. A sharp decrease in flow stress is found at 300 °C. The Khan-Liu (KL) model is modified to correlate with the measured thermo-mechanical responses of the two tempers over the studied, wide range of strain rates and temperatures. There is a close correlation between simulated and observed results.

Discovery of 7-hydroxyaporphines as conformationally restricted ligands for beta-1 and beta-2 adrenergic receptors.

A series of (-)-nornuciferidine derivatives was synthesized and the non-natural enantiomer of the aporphine alkaloid was discovered to be a potent ß1- and ß2-adrenergic receptor ligand that antagonized isoproterenol and procaterol induced cyclic AMP increases from adenylyl cyclase, respectively. Progressive deconstruction of the tetracyclic scaffold to less complex cyclic and acyclic analogues revealed that the conformationally restricted (6a-R,7-R)-7-hydroxyaporphine 2 (AK-2-202) was necessary for efficient receptor binding and antagonism.

Access to 6a-Alkyl Aporphines: Synthesis of (±)-N-Methylguattescidine.

(-)-N-Methylguattescidine (3) is an alkaloid recently isolated from Fissistigma latifolium and assigned as a rare example of a 6a-alkyl aporphine. Herein, we report the synthesis of (±)-3 and the des-hydroxyl derivative 4 using our previously reported ortho-phenol arylation methodology mediated by the XPhos precatalyst as a key synthetic step. In addition, substituents on the aryl halide portion of the ortho-phenol arylation substrates significantly influenced the formation of an oxidized side product.

Biopharmaceutic Profiling of Salts to Improve Absorption of Poorly Soluble Basic Drugs.

AZD1175 and AZD2207 are 2 highly lipophilic compounds with a significant risk of not achieving therapeutic plasma concentrations due to solubility-limited absorption. The compounds have the same molecular weight and minimal structural differences. The aim of the present work was to investigate whether salts could be applied to improve the intestinal absorption, and the subsequent in vivo exposure. Drug solubilities, dissolution rates, and degree of supersaturation and precipitation were determined in biorelevant media. Dog studies were performed, in the absence and presence of a precipitation inhibitor (hydroxypropyl methylcellulose). Finally, a human phase I study was performed. For AZD1175, there was a good agreement between dissolution rates, in vivo exposure in dog, and the obtained exposure in human with the selected hemi-1,5-naphthalenedisulfonate of the compound. For AZD2207, the picture was more complex. The same counter ion was selected for the study in man. In addition, the chloride salt of AZD2207 showed promising data in the presence of a precipitation inhibitor in vitro and in dog that, however, could not be repeated in man. The differences in observations between the 2 compounds could be attributed to the difference in solubility and to the degree of supersaturation in the gastric environment rather than in the intestine.

Synthetic studies of 7-oxygenated aporphine alkaloids: preparation of (-)-oliveroline, (-)-nornuciferidine, and derivatives.

7-Oxygenated aporphines 1-6 possessing anti-configurations have previously been reported. In order to explore their bioactivities, a synthesis was established by utilizing a diastereoselective reductive acid-mediated cyclization followed by palladium-catalyzed ortho-arylations. Moderate XPhos precatalyst loading (10 mol %) and short reaction times (30 min) were sufficient to mediate the arylations. Alkaloids 1-5 were successfully prepared, while (-)-artabonatine A was revised to syn-isomer 30. Consequently, (-)-artabonatine E likely also has a syn-configuration (31).

Regioselective synthesis of di-C-glycosylflavones possessing anti-inflammation activities.

Three methods are utilized to synthesize a variety of 6,8-di-C-glycosylflavones bearing identical or distinct glycosyl moieties. Some C-glycosylation compounds are found to have better anti-inflammation activities than the parent flavones. Among them, 6,8-di-C-glucosylapigenin (known as vicenin-2) shows inhibition of TNF-alpha expression and NO production with IC(50) values of 6.8 and 5.2 muM, respectively.

6,8-Di-C-glycosyl flavonoids from Dendrobium huoshanense.

Dendrobium huoshanense is a valued herbal plant used in traditional Chinese medicine. Fractionation of the water-soluble part of D. huoshanense by repeated chromatography culminated in the isolation of four new 6,8-di-C-glycosyl flavones (1-4), in addition to seven known compounds, comprising malic acid, dimethyl malate, N-phenylacetamide, isopentyl butyrate, salicylic acid, shikimic acid, and isoschaftoside. By detailed spectroscopic analysis, the structures of 1-4 were determined to have a core of apigenin bearing pentoside (arabinoside or xyloside) and rhamnosyl-hexoside (glucoside or galactoside) substituents.