Synthesis and Evaluation of Radamide Analogues, A Chimera of Radicicol and Geldanamycin

Previously, we reported the Hsp90 inhibitory activity of radamide, an open chain amide chimera of geldanamycin and radicicol. Attempts to further expand upon structure–activity relationships for this class of Hsp90 inhibitors led to the preparation of a series of radamide analogues focused on differing tether lengths and quinone mimics. In addition, the cup-shaped conformation adopted by the two natural products when bound to the Hsp90 N-terminal ATP binding pocket suggests that conformationally biased compounds may demonstrate improved binding and inhibition. The preparation and evaluation of radamide analogues with cis/trans α,β-unsaturated amides yielded compounds that exhibit improved antiproliferative activity. In addition, several analogues demonstrated the ability to induce degradation of Hsp90-dependent oncogenic signaling proteins in vitro, a hallmark of Hsp90 N-terminal inhibition

Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability

REV1 Inhibition Enhances Radioresistance and Autophagy

SIMPLE SUMMARY: Cancer resistance to therapy continues to be the biggest challenge in treating patients. Targeting the mutagenic translesion synthesis (TLS) polymerase REV1 was previously shown to sensitize cancer cells to chemotherapy. In this study, we tested the ability of REV1 inhibitors to radiation therapy and observed a lack of radiosensitization. In addition, we observed REV1 inhibition to trigger an autophagy stress response. Because reduction of REV1 triggered autophagy and failed to radiosensitize cells, we hypothesize REV1 expression dynamics might link cancer cell response to radiation treatment through the potential induction of autophagy. ABSTRACT: Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1’s important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens

Structure–Activity Relationships for Side Chain Oxysterol Agonists of the Hedgehog Signaling Pathway

Oxysterols (OHCs) are byproducts of cholesterol oxidation that are known to activate the Hedeghog (Hh) signaling pathway. While OHCs that incorporate hydroxyl groups throughout the scaffold are known, those that act as agonists of Hh signaling primarily contain a single hydroxyl on the alkyl side chain. We sought to further explore how side chain hydroxylation patterns affect oxysterol-mediated Hh activation, by performing a structure–activity relationship study on a series of synthetic OHCs. The most active analogue, 23­(<i>R</i>)-OHC (<b>35</b>), demonstrated potent activation of Hh signaling in two Hh-dependent cell lines (EC₅₀ values 0.54–0.65 μM). In addition, OHC <b>35</b> was approximately 3-fold selective for the Hh pathway as compared to the liver X receptor, a nuclear receptor that is also activated by endogenous OHCs. Finally, <b>35</b> induced osteogenic differentiation and osteoblast formation in cultured cells, indicating functional agonism of the Hh pathway

Identification of Vitamin D3-Based Hedgehog Pathway Inhibitors That Incorporate an Aromatic A‑Ring Isostere

Previous structure–activity relationship studies for vitamin D3 (VD3) inhibition of Hedgehog (Hh) signaling directed the design, synthesis, and evaluation of a series of VD3-based analogues that contain an aromatic A-ring mimic. Characterization of these compounds in a series of cellular assays demonstrated their ability to potently and selectively down-regulate Hh pathway signaling. The most active of these, <b>17</b>, inhibited pathway signaling in Hh-dependent mouse fibroblasts (IC₅₀ = 0.74 ± 0.1 μM) and cultured cancer cells (IC₅₀ values 3.8 ± 0.1 to 5.2 ± 0.2 μM). In addition, <b>17</b> demonstrated reduced activation of the vitamin D receptor (VDR) compared to VD3 in these cellular models. These results suggest that VD3-based analogues with an aromatic A-ring are a valid scaffold for the development of more selective and potent Hh pathway inhibitors and identify <b>17</b> as an intriguing lead from this class of compounds for further development. In addition, our analysis of Hh pathway inhibitors in cancer cells suggests that the murine basal cell carcinoma cell line ASZ001 and the human medulloblastoma cell line DAOY are appropriate in vitro cancer models for early stage evaluation of pathway inhibition

HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy

Virtual Pharmacophore Screening Identifies Small‐Molecule Inhibitors of the Rev1‐CT/RIR Protein–Protein Interaction

© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein–protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1-CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure-based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti-TLS potential