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Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class. A chemical screen identified BET bromodomain inhibitors as promoters of keratinocyte regenerative function and skin wound healing. Specifically, low-dose transient treatment with BET inhibitors imposes an activated, migratory state in keratinocytes.

Although FPPS is a potential anti-cancer target, the high bone affinity of nitrogen-containing bisphosphonates, FPPS inhibitors used clinically to treat bone disease, has prevented their development as cancer therapeutics. Using fragment-based drug discovery, non-bisphosphonate inhibitors were discovered that bind in a previously undescribed allosteric pocket. Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.

Dual orexin receptor (OXR) antagonists (DORAs) such as almorexant, SB-649868, suvorexant (MK-4305), and filorexant (MK-6096), have shown promise for the treatment of insomnias and sleep disorders. Whether antagonism of both OX1R and OX2R is necessary for sleep induction has been a matter of some debate. Experiments using knockout mice suggest that it may be sufficient to antagonize only OX2R. The recent identification of an orally bioavailable, brain penetrant OX2R preferring antagonist 2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one (IPSU) has allowed us to test whether selective antagonism of OX2R may also be a viable strategy for induction of sleep. We previously demonstrated that IPSU and suvorexant increase sleep when dosed during the mouse active phase (lights off); IPSU inducing sleep primarily by increasing NREM sleep, suvorexant primarily by increasing REM sleep. Here, our goal was to determine whether suvorexant and IPSU affect sleep architecture independently of overall sleep induction. We therefore tested suvorexant (25 mg/kg) and IPSU (50 mg/kg) in mice during the inactive phase (lights on) when sleep is naturally more prevalent and when orexin levels are normally low. Whereas IPSU was devoid of effects on the time spent in NREM or REM, suvorexant substantially disturbed the sleep architecture by selectively increasing REM during the first 4 h after dosing. At the doses tested, suvorexant significantly decreased wake only during the first hour and IPSU did not affect wake time. These data suggest that OX2R preferring antagonists may have a reduced tendency for perturbing NREM/REM architecture in comparison with DORAs. Whether this effect will prove to be a general feature of OX2R antagonists vs. DORAs remains to be seen.

Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various \"dual\" orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [(3)H]-BBAC ((S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the \"dual\" antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the \"dual\" antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species 1 . The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family 2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation 3 – 5 . Because SUCNR1 senses succinate as an immunological danger signal 6 —which has relevance for diseases including ulcerative colitis, liver fibrosis 7 , diabetes and rheumatoid arthritis 3 , 8 —it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo. High-resolution crystal structures of the rat succinate receptor SUCNR1 in an inactive confirmation, and the humanized rat SUCNR1 bound to an antagonist, provide insights into the structure of these receptors and the species selectivity of antagonist binding.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.sup.1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family.sup.2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation.sup.3-5. Because SUCNR1 senses succinate as an immunological danger signal.sup.6--which has relevance for diseases including ulcerative colitis, liver fibrosis.sup.7, diabetes and rheumatoid arthritis.sup.3,8--it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.sup.1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family.sup.2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation.sup.3-5. Because SUCNR1 senses succinate as an immunological danger signal.sup.6--which has relevance for diseases including ulcerative colitis, liver fibrosis.sup.7, diabetes and rheumatoid arthritis.sup.3,8--it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.sup.1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family.sup.2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation.sup.3-5. Because SUCNR1 senses succinate as an immunological danger signal.sup.6--which has relevance for diseases including ulcerative colitis, liver fibrosis.sup.7, diabetes and rheumatoid arthritis.sup.3,8--it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

RAS proteins control cell proliferation and activating mutations are collectively the most frequent oncogenic event observed in cancer patients, justifying investments into multiple drug discovery efforts. While RAS-directed therapeutic agents targeting either the inactive GDP-bound or the active GTP-bound state have entered the clinic, invariably resistance is observed. Mutations at drug binding sites represent a common resistance mechanism indicating the need to discover new targetable pockets in RAS. Such efforts are hindered by the small globular size of the protein, for long considered undruggable. Here we perform macrocyclic peptides mRNA and nanobody yeast display screens and discover a targetable ligand-induced pocket in RAS. In vitro and cellular experiments with the KM12 and KM12-AM nanobodies show RAS inhibition via displacement of cRAF, by affecting their protein-protein interaction via the less studied cRAF CRD domain. Further, we provide orthogonal functional validation for the discovered binding pocket via mutagenesis experiments. Notably, the discovered RAS-targeting approach enables simultaneous targeting of both GTP-bound active and GDP-bound inactive states and leaves the SwII pocket unaltered, opening possibilities of combinatorial approaches with clinically approved SwII pocket inhibitors. In this work, Beyer and colleagues have utilized display screening technologies to comprehensively chart RAS proteins “druggability” and in doing so unravel a targetable ligand-induced pocket in RAS opening unprecedented anti-RAS targeted opportunities.