Rational Design of Highly Potent, Selective, and Bioavailable SGK1 Protein Kinase Inhibitors for the Treatment of Osteoarthritis.

The serine/threonine kinase SGK1 is an activator of the ß-catenin pathway and a powerful stimulator of cartilage degradation that is found to be upregulated under genomic control in diseased osteoarthritic cartilage. Today, no oral disease-modifying treatments are available and chronic treatment in this indication sets high requirements for the drug selectivity, pharmacokinetic, and safety profile. We describe the identification of a highly selective druglike 1H-pyrazolo[3,4-d]pyrimidine SGK1 inhibitor 17a that matches both safety and pharmacokinetic requirements for oral dosing. Rational compound design was facilitated by a novel hSGK1 co-crystal structure, and multiple ligand-based computer models were applied to guide the chemical optimization of the compound ADMET and selectivity profiles. Compounds were selected for subchronic proof of mechanism studies in the mouse femoral head cartilage explant model, and compound 17a emerged as a druglike SGK1 inhibitor, with a highly optimized profile suitable for oral dosing as a novel, potentially disease-modifying agent for osteoarthritis.

Benchmarking computational methods for B-cell receptor reconstruction from single-cell RNA-seq data.

Multiple methods have recently been developed to reconstruct full-length B-cell receptors (BCRs) from single-cell RNA sequencing (scRNA-seq) data. This need emerged from the expansion of scRNA-seq techniques, the increasing interest in antibody-based drug development and the importance of BCR repertoire changes in cancer and autoimmune disease progression. However, a comprehensive assessment of performance-influencing factors such as the sequencing depth, read length or number of somatic hypermutations (SHMs) as well as guidance regarding the choice of methodology is still lacking. In this work, we evaluated the ability of six available methods to reconstruct full-length BCRs using one simulated and three experimental SMART-seq datasets. In addition, we validated that the BCRs assembled in silico recognize their intended targets when expressed as monoclonal antibodies. We observed that methods such as BALDR, BASIC and BRACER showed the best overall performance across the tested datasets and conditions, whereas only BASIC demonstrated acceptable results on very short read libraries. Furthermore, the de novo assembly-based methods BRACER and BALDR were the most accurate in reconstructing BCRs harboring different degrees of SHMs in the variable domain, while TRUST4, MiXCR and BASIC were the fastest. Finally, we propose guidelines to select the best method based on the given data characteristics.

The phospholipid-binding protein SESTD1 is a novel regulator of the transient receptor potential channels TRPC4 and TRPC5.

TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential cation channel family that have been implicated in important physiological functions, such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We therefore searched a human aortic cDNA library for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies, we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca(2+)-dependent. Because TRPC4 and -5 conduct Ca(2+), SESTD1-channel signaling may be bidirectional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins, such as SESTD1, adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction.

High throughput screening of mitochondrial bioenergetics in human differentiated myotubes identifies novel enhancers of muscle performance in aged mice.

Mitochondrial dysfunction is increasingly recognized as a contributor to age-related muscle loss and functional impairment. Therefore, we developed a high throughput screening strategy that enabled the identification of compounds boosting mitochondrial energy production in a human skeletal muscle cell model. Screening of 7949 pure natural products revealed 22 molecules that significantly increased oxygen consumption and ATP levels in myotubes. One of the most potent compounds was the flavanone hesperetin. Hesperetin (10 µM) increased intracellular ATP by 33% and mitochondrial spare capacity by 25%. Furthermore, the compound reduced oxidative stress in primary myotubes as well as muscle tissue in vivo. In aged mice administration of hesperetin (50 mg/kg/d) completely reverted the age-related decrease of muscle fiber size and improved running performance of treated animals. These results provide a novel screening platform for the discovery of drugs that can improve skeletal muscle function in patients suffering from sarcopenia or other disorders associated with mitochondrial dysfunction.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors.

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids.

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.

Formation of novel TRPC channels by complex subunit interactions in embryonic brain.

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.

[Establishment of an in vitro model system for the differentiation of synaptically coupled neurons from mouse embryonic stem cells]

The investigation of fundamental differentiation processes and toxic effects during the neurogenesis in the embryo is still difficult, since there are only a few suitable cellular model systems available so far. The isolation of neurons from early embryonic origin results in a very small amount of cells and a high number of laboratory animals is required for this purpose. Permanent tumor cell lines, able to differentiate into neuron-like cell types, are only a limited alternative for the use of primary cultures. None of the known permanent cell lines is capable to form terminally differentiated, synaptically coupled neurons in vitro. Embryonic stem (ES) cells can generate cell types of all 3 germ layers and provide a new possibility to study developmental processes on a cellular level. Nevertheless the successful differentiation of neurons from ES cells has not been shown. By using the ES cell line BLC 6 and cultivation of the cells in embryo-like aggregates (embryoid bodies), we were able to differentiate efficiently neuronal cells in vitro. The electrophysiological characterization of the BLC 6-derived neurons shows that these cells carry the complex electrical properties of postmitotic neurons and are coupled by inhibitory and excitatory synapses. The BLC 6 model probably represents the only developmental cell model suitable for studies from pluripotent stem cells to terminal differentiated neurons.

Real-time imaging of nuclear permeation by EGFP in single intact cells.

The NPC is the portal for the exchange of proteins, mRNA, and ions between nucleus and cytoplasm. Many small molecules (<10 kDa) permeate the nucleus by simple diffusion through the pore, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. In isolated Xenopus oocyte nuclei, diffusion of intermediate-sized molecules appears to be regulated by the NPC, dependent upon [Ca(2+)] in the nuclear envelope. We have applied real-time imaging and fluorescence recovery after photobleaching to examine the nuclear pore permeability of 27-kDa EGFP in single intact cells. We found that EGFP diffused bidirectionally via the NPC across the nuclear envelope. Although diffusion is slowed approximately 100-fold at the nuclear envelope boundary compared to diffusion within the nucleus or cytoplasm, this delay is expected for the reduced cross-sectional area of the NPCs. We found no evidence for significant nuclear pore gating or block of EGFP diffusion by depletion of perinuclear Ca(2+) stores, as assayed by a nuclear cisterna-targeted Ca(2+) indicator. We also found that EGFP exchange was not altered significantly during the cell cycle.

Pharmacophore-based search, synthesis, and biological evaluation of anthranilic amides as novel blockers of the Kv1.5 channel.

The search for novel, potent Kv1.5 blockers based on an anthranilic amide scaffold employing a pharmacophore-based virtual screening approach is described. The synthesis and structure-activity relationships (SAR) with respect to inhibition of the Kv1.5 channel are discussed. The most potent compounds display sub-micromolar inhibition of Kv1.5 and no significant effect on the HERG channel. In addition, good oral bioavailability is demonstrated for compound 3i in rats.