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The multi-receptor tyrosine kinase inhibitor XL092 has been developed to inhibit the activity of oncogenic targets, including MET, VEGFR2, and the TAM family of kinases TYRO3, AXL and MER. Presented here is a preclinical evaluation of XL092. XL092 causes a significant decrease in tumor MET and AXL phosphorylation (P < 0.01) in murine Hs 746T xenograft models relative to vehicle, and a 96% inhibition of VEGFR2 phosphorylation in murine lungs. Dose-dependent tumor growth inhibition with XL092 was observed in various murine xenograft models, with dose-dependent tumor regression seen in the NCI-H441 model. Tumor growth inhibition was enhanced with the combination of XL092 with anti-PD-1, anti-programmed death ligand-1 (PD-L1), or anti-CTLA-4 compared with any of these agents alone in the MC38 murine syngeneic model and with anti-PD-1 in the CT26 colorectal cancer survival model. In vivo, XL092 promoted a decrease in the tumor microvasculature and significant increases of peripheral CD4+ T cells and B cells and decreases in myeloid cells versus vehicle. Significant increases in CD8+ T cells were also observed with XL092 plus anti-PD-1 or anti-PD-L1 versus vehicle. In addition, XL092 promoted M2 to M1 repolarization of macrophages in vitro and inhibited primary human macrophage efferocytosis in a dose-dependent manner. In summary, XL092 was shown to have significant antitumor and immunomodulatory activity in animal models both alone and in combination with immune checkpoint inhibitors, supporting its evaluation in clinical trials., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding., Results: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals., Conclusions: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway., (© 2021. The Author(s).)

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo , which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a high-throughput screen (HTS). An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2 inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structure-activity relationships. The optimized compound 21 is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3-dioxygenase 1 (IDO1) and with improved human whole blood stability., Competing Interests: The authors declare no competing financial interest.

Background: A limited number of approved therapeutic options are available to metastatic medullary thyroid cancer (MTC) patients, and the response to conventional chemotherapy and/or radiotherapy strategies is inadequate. Sporadic and inherited mutations in the tyrosine kinase RET result in oncogenic activation that is associated with the pathogenesis of MTC. Cabozantinib is a potent inhibitor of MET, RET, and vascular endothelial factor receptor 2 (VEGFR2), as well as other tyrosine kinases that have been implicated in tumor development and progression. The object of this study was to determine the in vitro biochemical and cellular inhibitory profile of cabozantinib against RET, and in vivo antitumor efficacy using a xenograft model of MTC., Methods: Cabozantinib was evaluated in biochemical and cell-based assays that determined the potency of the compound against wild type and activating mutant forms of RET. Additionally, the pharmacodynamic modulation of RET and MET and in vivo antitumor activity of cabozantinib was examined in a MTC tumor model following subchronic oral administration., Results: In biochemical assays, cabozantinib inhibited multiple forms of oncogenic RET kinase activity, including M918T and Y791F mutants. Additionally, it inhibited proliferation of TT tumor cells that harbor a C634W activating mutation of RET that is most often associated with MEN2A and familial MTC. In these same cells grown as xenograft tumors in nude mice, oral administration of cabozantinib resulted in dose-dependent tumor growth inhibition that correlated with a reduction in circulating plasma calcitonin levels. Moreover, immunohistochemical analyses of tumors revealed that cabozantinib reduced levels of phosphorylated MET and RET, and decreased tumor cellularity, proliferation, and vascularization., Conclusions: Cabozantinib is a potent inhibitor of RET and prevalent mutationally activated forms of RET known to be associated with MTC, and effectively inhibits the growth of a MTC tumor cell model in vitro and in vivo.

Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Purpose: Acidic organelles, including Golgi bodies and lysosomes, are known to operate as Ca(2+) storage sites in many cell types. This study demonstrates the presence of Ca(2+) stores in lysosomes of bovine corneal endothelial cells (BCECs) and examines their interaction with Ins(1,4,5)P(3)-sensitive Ca(2+) stores., Methods: Glycyl-L-phenylalanine-beta-naphthylamide (GPN) was used to release Ca(2+) from lysosomes by inducing their selective osmotic swelling. Ca(2+) released into the cytoplasm was measured with fura-2 or fura-PE3 fluorescent dyes. Fluorescence of acridine orange (AO), which selectively sequesters into acidic organelles, was used to establish swelling of lysosomes in response to GPN., Results: Exposure to GPN (100-200 microM) in cultured BCECs produced an increase in free cytosolic Ca(2+) ([Ca(2+)](i)) equivalent to approximately 79% of the peak response to uridine triphosphate (UTP), a P2Y agonist (n = 19). The endothelium of the freshly isolated cornea also produced [Ca(2+)](i) transients similar to those in cultured BCECs; however, the peak [Ca(2+)](i) increase was smaller ( approximately 43% of the peak response to UTP; n = 13). In cultured BCECs, the response to UTP was unaffected by pretreatment with GPN with extracellular calcium ([Ca(2+)](o)) at 0 and 1.2 mM (n = 10). Neither pretreatment with thapsigargin (5 microM) nor with U73122 (a phospholipase C inhibitor; 10 microM) blocked the peak GPN response (n = 6). Exposure to 20 microM monensin produced a [Ca(2+)](i) increase with [Ca(2+)](o) at 0 and 1.2 mM and also reduced the subsequent peak response to GPN (n = 6)., Conclusions: GPN-sensitive lysosomal Ca(2+) stores, distinct from Ins(1,4,5)P(3)-sensitive Ca(2+) stores, are found in both cultured cells and fresh tissue. These stores are susceptible to depletion by the loss of the pH gradient across lysosomes and P2 agonists. The latter occurs through mechanisms independent of phospholipase C (PLC) activation or Ins(1,4,5)P(3). The GPN stores also induce [Ca(2+)](o) influx in response to their depletion.