Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

8-Hydroxyquinoline derivatives suppress GLI1-mediated transcription through multiple mechanisms.

Aberrant activation of the Hedgehog (Hh) signaling pathway has been observed in various human malignancies. Glioma-associated oncogene transcription factor 1 (GLI1) is the ultimate effector of the canonical Hh pathway and has also been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. The anti-cancer potential of GLI1 antagonism with small molecule inhibitors has demonstrated initial promise; however, the continued development of GLI1 inhibitors is still needed. We previously identified a scaffold containing an 8-hydroxyquinoline as a promising lead GLI1 inhibitor (compound 1). To further develop this scaffold, we performed a systematic structure-activity relationship study to map the structural requirements of GLI1 inhibition by this chemotype. A series of biophysical and cellular experiments identified compound 39 as an enhanced GLI1 inhibitor with improved activity. In addition, our studies on this scaffold suggest a potential role for SRC family kinases in regulating oncogenic GLI1 transcriptional activity.

Correction to "An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections".

[This corrects the article DOI: 10.1021/acscentsci.2c00598.].

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections.

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.

Structure-based virtual screening identifies an 8-hydroxyquinoline as a small molecule GLI1 inhibitor.

The glioma-associated family of transcription factors (GLI) have emerged as a promising therapeutic target for a variety of human cancers. In particular, GLI1 plays a central role as a transcriptional regulator for multiple oncogenic signaling pathways, including the hedgehog (Hh) signaling pathway. We undertook a computational screening approach to identify small molecules that directly bind GLI1 for potential development as inhibitors of GLI-mediated transcription. Through these studies, we identified compound 1, which is an 8-hydroxyquinoline, as a high-affinity binder of GLI1. Compound 1 inhibits GLI1-mediated transcriptional activity in several Hh-dependent cellular models, including a primary model of murine medulloblastoma. We also performed a series of computational analyses to define more clearly the mechanism(s) through which 1 inhibits GLI1 function after binding. Our results strongly suggest that binding of 1 to GLI1 does not prevent GLI1/DNA binding nor disrupt the GLI1/DNA complex, but rather, it induces specific conformational changes in the overall complex that prevent proper GLI function. These results highlight the potential of this compound for further development as an anti-cancer agent that targets GLI1.

Structure-Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis.

Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1-CT PPIs with Rev1-interacting regions (RIRs) of TLS polymerases. To explore the structure-activity relationships for this scaffold, we developed a protocol for co-crystallization of compounds that target the RIR binding site on Rev1-CT with a triple Rev1-CT/Rev7R124A /Rev3-RBM1 complex, and solved an X-ray crystal structure of Rev1-CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1-CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1-CT by microscale thermophoresis (MST). Several second-generation PAP derivatives showed an affinity for Rev1-CT that was improved by over an order of magnitude, thereby validating the structure-based assumptions that went into the compound design.

Exploring the physicochemical and antiproliferative properties of biaryl-linked [13]-macrodilactones.

A macrocyclic motif fosters productive protein-small molecule interactions. There are numerous examples of both natural product and designed, synthetic macrocycles that modulate the immune system, slow microbial infection, or kill eukaryotic cells. Reported here are the synthesis, physicochemical characterization, and antiproliferative activity of a group of [13]-macrodilactones decorated with a pendant biaryl moiety. Biaryl analogs were prepared by Suzuki reactions conducted on a common intermediate that contained a bromophenyl unit alpha to one of the carbonyls of the [13]-macrodilactone. Principal component analysis placed the new compounds in physicochemical context relative to a variety of pharmaceuticals and natural products. Modest inhibition of proliferation was observed in ASZ cells, a murine basal cell carcinoma line. This work underscores the value of an approach toward the identification of bioactive compounds that places the evaluation of physicochemical parameters early in the search process.

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.

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.

Probing the Protein-Protein Interaction Between the ATRXADD Domain and the Histone H3 Tail.

While loss-of-function mutations in the ATRX gene have been implicated as a driving force for a variety of pediatric brain tumors, as well as pancreatic neuroendocrine tumors, the role of ATRX in gene regulation and oncogenic development is not well-characterized. The ADD domain of ATRX (ATRXADD) localizes the protein to chromatin by specifically binding to the histone H3 tail. This domain is also a primary region that is mutated in these cancers. The overall goal of our studies was to utilize a variety of techniques (experimental and computational) to probe the H3:ATRXADD protein-protein interaction (PPI). We developed two biochemical assays that can be utilized to study the interaction. These assays were utilized to experimentally validate and expand upon our previous computational results. We demonstrated that the three anchor points in the H3 tail (A1, K4, and K9) are all essential for high affinity binding and that disruption of more than one contact region will be required to develop a small molecule that disrupts the PPI. Our approach in this study could be applied to other domains of ATRX, as well as PPIs between other distinct proteins.

Structure-Activity Relationships for Itraconazole-Based Triazolone Analogues as Hedgehog Pathway Inhibitors.

The Food and Drug Administration-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and has the potential to serve as an anticancer chemotherapeutic against several Hh-dependent malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through the design, synthesis, and evaluation of novel des-triazole ITZ analogues that incorporate modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that the triazolone/side chain region can be replaced with various functionalities (hydrazine carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ. Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for both potent Hh pathway inhibition and compound stability. Finally, our studies suggest that the ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further improve its anti-Hh potency and physicochemical properties.

A metadynamic approach to understand the recognition mechanism of the histone H3 tail with the ATRXADD domain.

The binding affinity between the histone 3 (H3) tail and the ADD domain of ATRX (ATRXADD) increases with the subsequent addition of methyl groups on lysine 9 on H3. To improve our understanding of how the difference in methylation state affects binding between H3 and the ATRXADD, we adopted a metadynamic approach to explore the recognition mechanism between the two proteins and identify the key intermolecular interactions that mediate this protein-peptide interaction (PPI). The non-methylated H3 peptide is recognized only by the PHD finger of ATRXADD while mono-, di-, and trimethylated H3 is recognized by both the PHD and GATA-like zinc finger of the domain. Furthermore, water molecules play an important role in orienting the lysine 9 anchor towards the GATA-like zinc finger, which results in stabilizing the lysine 9 binding pocket on ATRXADD. We compared our computational results against experimentally determined NMR and X-ray structures by demonstrating the RMSD, order parameter S2 and hydration number of the complex. The metadynamics data provide new insight into roles of water-bridges and the mechanisms through which K9 hydration stabilizes the H3K9me3:ATRXADD PPI, providing context for the high affinity demonstrated between this protein and peptide.