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Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) is a serine/threonine kinase that acts as an immune checkpoint downstream of T-cell receptor stimulation. MAP4K1 activity is enhanced by prostaglandin E2 (PGE2) and transforming growth factor beta (TGFβ), immune modulators commonly present in the tumor microenvironment. Therefore, its pharmacological inhibition is an attractive immuno-oncology concept for inducing therapeutic T-cell responses in cancer patients. Here, we describe the systematic optimization of azaindole-based lead compound 1 , resulting in the discovery of potent and selective MAP4K1 inhibitor 38 (BAY-405) that displays nanomolar potency in biochemical and cellular assays as well as in vivo exposure after oral dosing. BAY-405 enhances T-cell immunity and overcomes the suppressive effect of PGE2 and TGFβ. Treatment of tumor-bearing mice shows T-cell-dependent antitumor efficacy. MAP4K1 inhibition in conjunction with PD-L1 blockade results in a superior antitumor impact, illustrating the complementarity of the single agent treatments.

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756)., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRAS G12C -SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC 50 of 21 nM and is a valuable chemical probe for future investigations., Competing Interests: Conflict of interest statement: R.C.H. B.S., J.S., D.M., A.H., C.M.S., N.D.W., H.B., U.B., J.W., V.B., J.M., K.P., G.S., N.B., K.E., K.G., L.W., F.v.N., and B.B. are or have been employees and stockholders of Bayer AG. J.D.K. and D.W. are employees of Evotec AG. R.C.H., B.S., J.S., D.M., H.B., K.P., N.B., K.E., L.W., F.v.N., and B.B. are coauthors of a patent application., (Copyright © 2019 the Author(s). Published by PNAS.)

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.

Unlabelled: Imaging of amyloid-β (Aβ) plaques by PET is more and more integrated into concepts for Alzheimer disease (AD) diagnosis and drug development. The objective of this study was to find novel chemical entities that can be transformed into (18)F-labeled Aβ tracers with favorable brain washout kinetics and low background signal., Methods: High-throughput screening of a large chemical library was used to identify new ligands for fibrillar aggregates of Aβ(1-42) peptide. Thirty-two fluorinated derivatives were synthesized and tested for their affinity toward AD brain homogenate. Twelve ligands have been radiolabeled with (18)F. The pharmacokinetic properties of the radioligands were investigated in mouse and monkey biodistribution studies. Binding characteristics were determined by autoradiography of AD brain sections in vitro and using amyloid precursor protein transgenic mice in vivo., Results: The systematic search for Aβ imaging agents revealed several fluorinated derivatives with nanomolar affinity for Aβ. The fluoropyridyl derivative BAY 1008472 showed a high initial brain uptake (6.45 percentage injected dose per gram at 2 min) and rapid brain washout (ratio of percentage of injected dose per gram of tissue at 2 and 30 min after injection, 9.2) in mice. PET studies of healthy rhesus monkeys confirmed the high initial brain uptake of BAY 1008472 (2.52 standardized uptake value at peak) and a fast elimination of total radioactivity from gray and white matter areas (ratio of standardized uptake value at peak uptake and 60 min 11.0). In autoradiographic analysis, BAY 1008472 selectively detected Aβ deposits in human AD brain sections with high contrast and did not bind to τ- or α-synuclein pathologies. Finally, ex vivo autoradiography of brain sections from amyloid precursor protein-transgenic mice confirmed that BAY 1008472 is indeed suitable for the in vivo detection of Aβ plaques., Conclusion: A new chemical class of Aβ tracers has been identified by high-throughput screening. The fluoropyridyl derivative BAY 1008472 shows a favorable preclinical profile including low background binding in gray and white matter. These properties might qualify this new tracer, in particular, to detect subtle amounts or changes of Aβ burden in presymptomatic AD and during therapy.

A crystallographic fragment screen was carried out to identify starting points for the development of inhibitors of protein kinase Pim-1, a potential target for tumour therapy. All fragment hits identified via soaking in this study turned out to bind to the unusually hydrophobic pocket at the hinge region. The most potent fragments, two cinnamic acid derivatives (with a best IC(50) of 130 µM), additionally form a well defined hydrogen bond. The balance between hydrophobic and polar interactions makes these molecules good starting points for further optimization. Pim-2 inhibitors from a recently reported high-throughput screening campaign also feature a cinnamic acid moiety. Two of these Pim-2 inhibitors were synthesized, their potencies against Pim-1 were determined and their cocrystal structures were elucidated in order to determine to what degree the binding modes identified by fragment screening are conserved in optimized inhibitors. The structures show that the cinnamic acid moieties indeed adopt the same binding mode. Fragment screening thus correctly identified binding modes which are maintained when fragments are grown into larger and higher affinity inhibitors. The high-throughput screening-derived compound (E)-3-{3-[6-(4-aminocyclohexylamino)-pyrazin-2-yl]phenyl}acrylic acid (compound 1) is the most potent inhibitor of the cinnamic acid series for which the three-dimensional binding mode is known (IC(50) = 17 nM, K(d) = 28 nM). The structure reveals the molecular basis for the large gain in potency between the initial fragment hit and this optimized inhibitor.

Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.