Optimization of N-Piperidinyl-Benzimidazolone Derivatives as Potent and Selective Inhibitors of 8-Oxo-Guanine DNA Glycosylase 1.

8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : C→T : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α-hydroxyketones.

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation.

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

Targeting BER enzymes in cancer therapy.

Base excision repair (BER) repairs mutagenic or genotoxic DNA base lesions, thought to be important for both the etiology and treatment of cancer. Cancer phenotypic stress induces oxidative lesions, and deamination products are responsible for one of the most prevalent mutational signatures in cancer. Chemotherapeutic agents induce genotoxic DNA base damage that are substrates for BER, while synthetic lethal approaches targeting BER-related factors are making their way into the clinic. Thus, there are three strategies by which BER is envisioned to be relevant in cancer chemotherapy: (i) to maintain cellular growth in the presence of endogenous DNA damage in stressed cancer cells, (ii) to maintain viability after exogenous DNA damage is introduced by therapeutic intervention, or (iii) to confer synthetic lethality in cancer cells that have lost one or more additional DNA repair pathways. Here, we discuss the potential treatment strategies, and briefly summarize the progress that has been made in developing inhibitors to core BER-proteins and related factors.

Multi-step biocatalytic strategies for chiral amino alcohol synthesis.

Chiral amino alcohols are structural motifs present in sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Their chemical synthesis presents several challenges in establishing at least two chiral centres. Here a de novo metabolic pathway using a transketolase enzyme coupled with a transaminase enzyme has been assembled. To synthesise this motif one of the strategies to obtain high conversions from the transaminase/transketolase cascade is the use of hydroxypyruvate (HPA) as a two-carbon donor for the transketolase reaction; although commercially available it is relatively expensive limiting application of the pathway on an industrial scale. Alternately, HPA can be synthesised but this introduces a further synthetic step. In this study two different biocatalytic strategies were developed for the synthesis of (2S,3R)-2-amino-1,3,4-butanetriol (ABT) without adding HPA into the reaction. Firstly, a sequential cascade of three enzymatic steps (two transaminases and one transketolase) for the synthesis of ABT from serine, pyruvate and glycolaldehyde as substrates. Secondly, a two-step recycling cascade where serine is used as donor to aminate erythrulose (catalysed by a transketolase) for the simultaneous synthesis of ABT and HPA. In order to test the novel pathways, three new transaminases are described, two ω-transaminases able to accept a broad range of amine acceptors with serine as amine donor; and an α-transaminase, which showed high affinity towards serine (KM: 18mM) using pyruvate as amine acceptor. After implementation of the above enzymes in the biocatalytic pathways proposed in this paper, the two-step recycling pathway was found to be the most promising for its integration with E. coli metabolism. It was more efficient (10-fold higher conversion), more sustainable and cost-effective (use of low cost natural substrates and only two enzymes), and the reaction could be performed in a one-pot system.