The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that ensures cellular proteostasis by maintaining the folding, stabilization, activation, and degradation of over 400 client proteins. Hsp90 is not only critical for routine protein maintenance in healthy cells, but also during states of cellular stress, such as cancer and neurodegenerative diseases. Due to its ability to affect phosphorylation of numerous client proteins, inhibition of Hsp90 has been an attractive anticancer approach since the early 1990’s, when researchers identified a druggable target on the amino terminus of Hsp90 for a variety of cancers. Since then, 17 Hsp90 inhibitors that target the chaperone’s Nterminal domain, have entered clinical trials. None, however, have been approved thus far by the FDA as a cancer monotherapy. In these trials, a major limitation observed with Hsp90 inhibition at the N-terminal domain was dose-limiting toxicities and relatively poor pharmacokinetic profiles. Despite this, preclinical and clinical research continues to show that Hsp90 inhibitors effectively target cancer cell death and decrease tumor progression supporting the rationale for the development of novel Hsp90 inhibitors. Here, we present an in-depth overview of the Hsp90 inhibitors used in clinical trials. Finally, we present current shifts in the field related to targeting the carboxy-terminal domain of Hsp90 as well as to the development of isoform-selective inhibitors as a means to bypass the pitfalls of current Hsp90 inhibitors and improve clinical trial outcomes.

Herein, we report the SAR leading to the discovery of VU6028418, a potent M 4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1‑4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

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High grade gliomas (HGG), including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), carry poor prognosis with median survival rates under 15 months post diagnosis. Due to dysregulation of the RTK/PI3K pathways within these tumors, including RTK pathway amplifications, targeted kinase inhibition has been considered a promising strategy to improve patient outcomes. However, many single-agent inhibitors of EGFR or PI3K have shown limited response in gliomas due to poor blood-brain barrier (BBB) penetrance and activation of compensatory signaling. The design of BBB-penetrant novel therapeutics that target multiple kinases is crucial for overcoming these obstacles. Single-molecule multikinase inhibitors may decrease resistance, present a single pharmacokinetic dosage profile, and reduce risks of multi-agent toxicities, supporting this strategy over dual drug approaches. A panel of inhibitors exploiting known binding modes of structurally-related ATP binding site inhibitors of EGFR/PI3K were synthesized and characterized, of which six promising candidates were chosen for in vitro study. The interaction of the inhibitors with common drug efflux proteins were probed to predict BBB penetrance. Of the compounds, MTX-241 was least likely to act as a substrate for efflux proteins such as P-glycoprotein. Treatment of MTX-241 in three human U87 GBM lines, some of which overexpress wild-type or vIII EGFR, three patient-derived DIPG lines, and two glioma stem cell (GSC) lines, one of which is radioresistant, showed strong cytotoxic potency measured by growth inhibitory activity, with IC50s in the <10 µM range. MTX-241 was significantly more potent than clinically relevant inhibitors targeting EGFR/RTKs (gefitinib, lapatinib, dasatinib, imatinib) or PI3K (alpelisib, idelalisib). Notably, selectivity of MTX-241 towards these HGG cell lines in comparison to normal human astrocytes (NHA) was observed, with NHA nearly insensitive to MTX-241 even at >100 µM. We found significant inhibition of p-EGFR and p-Akt in these cell lines compared to DMSO controls. Further, we identified a unique glycolytic-suppressing activity of MTX-241 in the U87/DIPG lines, to a greater extent than treatment with gefitinib or alpelisib. Additionally, as the clinically relevant HDAC inhibitor vorinostat upregulated EGFR activity, we examined the combination of MTX-241 and vorinostat, which significantly reduced proliferative abilities, achieving synergistic combination indices. MTX-241 and vorinostat treatment in the NHA model was not synergistic, highlighting the selectivity for HGG tumor models. Our data suggests that a dual inhibitor of EGFR and PI3K, alone or in combination with an HDAC inhibitor, represents a viable therapeutic strategy in adult/pediatric HGG. Future studies will focus on evaluation of in vivo efficacy in tumor-bearing mouse models. Citation Format: Yusha Y. Sun, Cavan P. Bailey, Trever R. Carter, Christopher E. Whitehead, Judith S. Sebolt-Leopold, Joya Chandra. Evaluation of highly potent and selective single-molecule dual EGFR/PI3K inhibitors in preclinical models of adult and pediatric high-grade glioma [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 3089.