Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Macitentan does not interfere with hepatic bile salt transport.

Treatment of pulmonary arterial hypertension with the endothelin receptor antagonist bosentan has been associated with transient increases in liver transaminases. Mechanistically, bosentan inhibits the bile salt export pump (BSEP) leading to an intrahepatic accumulation of cytotoxic bile salts, which eventually results in hepatocellular damage. BSEP inhibition by bosentan is amplified by its accumulation in the liver as bosentan is a substrate of organic anion-transporting polypeptide (OATP) transport proteins. The novel endothelin receptor antagonist macitentan shows a superior liver safety profile. Introduction of the less acidic sulfamide moiety and increased lipophilicity yield a hepatic disposition profile different from other endothelin receptor antagonists. Passive diffusion rather than OATP-mediated uptake is the driving force for macitentan uptake into the liver. Interaction with the sodium taurocholate cotransporting polypeptide and BSEP transport proteins involved in hepatic bile salt homeostasis is therefore limited due to the low intrahepatic drug concentrations. Evidence for this conclusion is provided by in vitro experiments in drug transporter-expressing cell lines, acute and long-term studies in rats and dogs, absence of plasma bile salt changes in healthy human volunteers after multiple dosing, and finally the liver safety profile of macitentan in the completed phase III morbidity/mortality SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary Arterial Hypertension to Improve Clinical Outcome) trial.

Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies.

Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prepare and incubate rat and human liver and intestinal slices. Slices are prepared from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O(2)/5% CO(2) atmosphere.

Lack of strain-related differences in drug metabolism and efflux transporter characteristics between CD-1 and athymic nude mice.

CD-1 mice are commonly used in oncology metabolism and toxicity to support drug discovery and development and to examine drug metabolism and toxicity properties of new chemical entities. On the other hand, athymic nude mice are the preferred animals to investigate tumor growth inhibition. Therefore, a frequently asked question is: are the metabolic and pharmacokinetic characteristics of xenobiotics in these two mouse strains comparable or not? To address this issue, we characterized drug metabolism and efflux transporter properties in both strains and in different organs. The metabolic stability of a set of 20 compounds and metabolite formation of cytochrome P450 (CYP) marker substrates (testosterone, ethoxyresorufin and pentoxyresorufin) were measured in liver microsomes. Drug conjugation was studied by following the disappearance of 7-hydroxycoumarin and the formation of its glucuronide and sulfate conjugates in freshly prepared liver slices. In addition, mRNA expression levels of the main cyp genes and drug efflux transporters were investigated by real-time RT-PCR in the liver, kidney, intestine and adrenal glands. No significant differences in enzymatic activities and metabolite formation were observed between the two strains. Also mRNA expression profiles of cyp and drug transporter genes were similar between CD-1 and nude mice.

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies.

INTRODUCTION: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. METHODS: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. RESULTS: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. DISCUSSION: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.

Phase I and phase II metabolic activities are retained in liver slices from mouse, rat, dog, monkey and human after cryopreservation.

Precision-cut liver slices are described as a valuable tool for in vitro metabolism studies of potential drug candidates. Recently, some papers reported successful cryopreservation conditions for liver slices, facilitating a broader and more efficient use of the tissue (particularly of human origin). The aim of this study is to evaluate the effect of cryopreservation on both phase I and phase II metabolism in liver slices prepared from mouse, rat, dog, monkey and human, using rapid freezing in the presence of 18% DMSO. Glucuronidation and sulfation activities (phase II) in both freshly prepared and cryopreserved liver slices were determined by rapid LC-MS/MS analyses using 7-hydroxycoumarin as a marker substrate. Testosterone was used as a marker substrate for cytochrome P450 mediated drug metabolism (phase I). Although the metabolic patterns and rates varied among the different species, the phase I and phase II metabolic capacities of the liver slices were well maintained after cryopreservation. Despite the good biotransformation capacity of cryopreserved slices a decrease in viability, expressed as ATP content and LDH leakage, was observed. MTT reduction was well maintained after cryopreservation. The possibility to cryopreserve liver slices will allow a more efficient utilisation of tissue, in particular from human, but also from dog and monkey. Finally, cryopreserved liver slices from mouse, rat, dog, monkey and human with good phase I and II metabolism activities are a useful in vitro tool to compare metabolite profiles of new chemical entities between species.