Xilio Therapeutics (United States)

Fermented wheat germ extract (FWGE), a nutraceutical with reported anticancer properties, contains numerous biologically active molecules, but its precise therapeutic constituents remain unclear. In this study, we identify and characterize a novel small-molecule inhibitor, CSH-4044, isolated from FWGE. Through preparative HPLC and structural elucidation via X-ray crystallography, CSH-4044 was revealed to be a unique benzothiazole compound. Kinase profiling demonstrated its high specificity toward the PIM and DYRK families of protein kinases. We determined the co-crystal structure of CSH-4044 bound to PIM1, revealing ATP-competitive binding, and critical hydrophobic and hydrogen-bonding interactions. A chemically synthesized version of CSH-4044 mirrored the activity of the natural product, confirming structural integrity and biological equivalence. Functionally, CSH-4044 suppressed PIM3-driven BAD phosphorylation in pancreatic cancer cells and reduced DYRK1A-mediated Tau phosphorylation in neuronal cells. Our findings position CSH-4044 as a promising lead for targeting PIM and DYRK families of kinases and highlight FWGE as a source of potential therapeutic compounds.

BackgroundInterleukin-12 (IL-12) is a proinflammatory cytokine that bridges innate and adaptive immunity by stimulating the differentiation of T helper 1 cells and enhancing the cytolytic activity of natural killer (NK) and T cells. Historically, IL-12 has demonstrated potent anti-tumor activity in syngeneic mouse models and promising efficacy in humans. However, development of IL-12-based agents has been limited by severe systemic toxicities in the clinical setting. We created XTX301, an investigational tumor-activated, half-life extended engineered IL-12, to reduce toxicity and improve the therapeutic index of IL-12. XTX301 masking domain is designed to pharmacologically inactivate IL-12 in non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage by proteases enriched in the tumor microenvironment (TME).MethodsWe created mXTX301, a murine surrogate for XTX301, and its unmasked control molecule for in vivo pharmacology assessments, as human IL-12 does not bind to mouse IL-12 receptors. We assessed the anti-tumor activity of mXTX301 in cancer mouse models with diverse immune phenotypes, including MC38, CT26, B16F10, and MB49. Flow cytometry and bulk RNA sequencing analysis were performed to elucidate the mechanism of action of mXTX301. XTX301 and mXTX301 cleavage was assessed ex vivo in human and mouse tumor samples. In addition, mXTX301 tumor specific activation was evaluated in the MC38 tumor model. mXTX301 anti-tumor activity in combination with immunotherapies including checkpoint blockade were also tested in a mouse model.ResultsmXTX301 was well-tolerated compared to the unmasked control molecule and resulted in anti-tumor activity across multiple syngeneic mouse tumor models with diverse immune phenotypes. mXTX301 also demonstrated efficacy in mice with large (~400 mm3) MC38 tumors that have highly immunosuppressive TME and typically respond poorly to treatment. mXTX301 anti-tumor activity was associated with increases in the IFN-γ cell signaling pathway and activation of NK and T cells. Tumor selective activation of XTX301 and mXTX301 was observed ex vivo in human tumor and plasma samples, and in vivo and ex vivo in mouse, respectively. Data in preclinical models also suggest the potential for XTX301 to be combined with other immunotherapies to improve anti-tumor activity.ConclusionsIn preclinical models, mXTX301, a tumor activated, half-life extended IL-12, demonstrated remodeling of the TME, anti-tumor activity, and improved tolerability compared to a systemically active IL-12, indicating that XTX301 has potential for activity in both hot and cold tumors while also improving the therapeutic index of IL-12 treatment. XTX301 is being evaluated in a Phase I clinical trial (NCT05684965).

In predisposed individuals, migraine evolves into a stage of daily or nearly daily headaches, known as chronic migraine. Although relatively prevalent and debilitating at all ages, chronic migraine is particularly aggressive in the pediatric population. Several risk factors for chronic migraine have been identified, largely due to two very large longitudinal studies, the American Migraine Prevalence and Prevention Study (AMPP) and the Attention Brazil Project (ABP). This review summarizes lessons learned from these studies that included children from 5 to 19 years of age. We start by contextualizing chronic migraine and by offering a systematic approach to diagnosis. We then discuss pre-natal and post-natal risk factors for migraine transformation, and close by reviewing treatment strategies, ultimately attempting to offer a meaningful overview of chronic migraine in pre-adults based on our experience conducting these studies.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.