Chemical Tools Research Articles

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Development of visible-light-activatable photocaged PROTACs

Photocaged proteolysis-targeting chimeras (PROTACs), which employ light as a stimulus to control protein degradation, have recently garnered considerable attention as both powerful chemical tools and a promising therapeutic strategy. However, the poor penetration depth of traditionally used ultraviolet light and the deficiency of alternative caging positions have restricted their applications in biological systems. By installing a diverse array of photocaged groups, with excitation wavelengths ranging from 365 nm to 405 nm, onto different positions of cereblon (CRBN) and Von Hippel-Lindau (VHL)-recruiting Brd4 degraders, we conducted the first comprehensive study on visible-light-activatable photocaged PROTACs to the best of our knowledge. We found the A2, A4 and B3 positions to be most effective at regulating the activity of the degraders, and to provide the resulting molecules (9–12 and 17) as potent visible-light-controlled degraders in live cells.

Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 μM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

Discovery and Development of Cyclic Peptide Proteasome Stimulators.

The proteasome degrades proteins, which is essential for cellular homeostasis. Ubiquitin independent proteolysis degrades highly disordered and misfolded proteins. A decline of proteasomal activity has been associated with multiple neurodegenerative diseases due to the accumulation of misfolded proteins. In this work, cyclic peptide proteasome stimulators (CyPPSs) that enhance the clearance of misfolded proteins were discovered. In the initial screen of predicted natural products (pNPs), several cyclic peptides were found to stimulate the 20S core particle (20S CP). Development of a robust structural activity relationship led to the identification of potent, cell permeable CyPPSs. In vitro assays revealed that CyPPSs stimulate degradation of highly disordered and misfolded proteins without affecting ordered proteins. Furthermore, using a novel flow-based assay for proteasome activity, several CyPPSs were found to stimulate the 20S CP in cellulo. Overall, this work describes the development of CyPPSs as chemical tools capable of stimulating the proteasome and provides strong support for proteasome stimulation as a therapeutic strategy for neurodegenerative diseases.

Discovery of a cannabinoid CB2 receptor fluorescent probe based on a pyridin-2-yl-benzyl-imidazolidine-2,4-dione scaffold

Cannabinoid receptor type 2 (CB2R) agonists have therapeutic potential for the treatment of (neuro)inflammatory diseases. Fluorescent probes enable the detection of CB2R in relevant cell types and serve as a chemical tool in cellular target engagement studies. Here, we report the structure-based design and synthesis of a new CB2R selective fluorescent probe. Based on the cryo-EM structure of LEI-102 in complex with the CB2R, we synthesized 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione analogues in which we introduced a variety of linkers and fluorophores. Molecular pharmacological characterization showed that compound 22 containing a Cy5-fluorophore with an alkyl-spacer was the most potent probe with a pKi of 6.2 ± 0.6. It was selective over the cannabinoid CB1 receptor and behaved as an inverse agonist (EC50 5.3 ± 0.1, Emax -63% ± 6). Probe 22 may serve as a chemical tool in target and lead validation studies for the CB2R.

Discovery of a far-red carbazole-benzoindolium fluorescent ligand that selectively targets mitochondrial DNA and suppresses breast cancer growth

G-quadruplex (G4) formation was considered to be more prevalent in the mitochondrial DNA (mtDNA) of cancer cells compared with normal cells. Stabilization of these G4s may induce mtDNA instability and cause mitochondrial dysfunction and subsequent cell death, which may be treated as a new strategy for cancer treatment. However, few ligands were developed to target mtG4s, leaving a huge room to improve. In this study, we designed and synthesized a series of carbazole-based ligands, among which, BKN-1 was identified as the most promising mitochondrial targeting fluorescent ligand with far-red emission. Then, we demonstrated that BKN-1 may robustly interact with mtG4s via a variety of biophysical, biological experiments. Subsequently, we proved that BKN-1 may cause mtDNA loss, disrupt mitochondrial integrity, decrease ATP level and trigger unbalanced ROS, thereby leading to apoptosis and autophagy. Finally, we verified that BKN-1 had good anti-tumor activity in both cellular and in vivo models. Altogether, this study provided a dual-function ligand that may not only track the formation of mtG4s but also induce mitochondrial dysfunction, which may be developed into an applicable chemical tool for investigating the structure and function of mtG4s, and moreover, an effective therapeutic agent for cancer interference.

Early Phases of Star Formation: Testing Chemical Tools

Star-forming processes strongly influence the ISM chemistry. Nowadays, many high-quality databases are available at millimeter wavelengths. Using them, it is possible to carry out studies that review and deepen previous results. If these studies involve large samples of sources, it is preferred to use direct tools to study the molecular gas. With the aim of testing these tools such as the use of the HCN/HNC ratio as a thermometer, and the use of H13CO+, HC3N, N2H+ and C2H as “chemical clocks,” we present a molecular line study toward 55 sources representing massive young stellar objects at different evolutionary stages: infrared dark clouds (IRDCs), high-mass protostellar objects (HMPOs), hot molecular cores (HMCs) and ultracompact H ii regions. We found that the use of the HCN/HNC ratio as a universal thermometer in the ISM should be taken with care because the HCN optical depth is a big issue that can affect the method. Hence, this tool should be utilized only after a careful analysis of the HCN spectrum, checking that no line, neither the main nor the hyperfine ones, presents absorption features. We point out that the analysis of the emission of H13CO+, HC3N, N2H+ and C2H could be useful to trace and distinguish regions among IRDCs, HMPOs and HMCs. The molecular line widths of these four species increase from the IRDC to the HMC stage, which can be a consequence of the gas dynamics related to the star-forming processes taking place in the molecular clumps. Our results not only contribute with more statistics, acting as a probe of such chemical tools, useful to obtain information in large samples of sources, but also complement previous works through the analysis of other types of sources.

Late-stage synthesis of heterobifunctional molecules for PROTAC applications via ruthenium-catalysed C‒H amidation

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules emerging as a powerful modality in drug discovery, with the potential to address outstanding medical challenges. However, the synthetic feasibility of PROTACs, and the empiric and complex nature of their structure-activity relationships continue to present formidable limitations. As such, modular and reliable approaches to streamline the synthesis of these derivatives are highly desirable. Here, we describe a robust ruthenium-catalysed late-stage C‒H amidation strategy, to access fully elaborated heterobifunctional compounds. Using readily available dioxazolone reagents, a broad range of inherently present functional groups can guide the C–H amidation on complex bioactive molecules. High selectivity and functional group tolerance enable the late-stage installation of linkers bearing orthogonal functional handles for downstream elaboration. Finally, the single-step synthesis of both CRBN and biotin conjugates is demonstrated, showcasing the potential of this methodology to provide efficient and sustainable access to advanced therapeutics and chemical biology tools.

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

Biocatalytic cyclization of small macrolactams by a penicillin-binding protein-type thioesterase.

Macrocyclic peptides represent promising scaffolds for chemical tools and potential therapeutics. Synthetic methods for peptide macrocyclization are often hampered by C-terminal epimerization and oligomerization, leading to difficult scalability. While chemical strategies to circumvent this issue exist, they often require specific amino acids to be present in the peptide sequence. Herein, we report the characterization of Ulm16, a peptide cyclase belonging to the penicillin-binding protein-type class of thioesterases that catalyze head-to-tail macrolactamization of nonribosmal peptides. Ulm16 efficiently cyclizes various nonnative peptides ranging from 4 to 6 amino acids with catalytic efficiencies of up to 3 × 106 M-1 s-1. Unlike many previously described homologs, Ulm16 tolerates a variety of C- and N-terminal amino acids. The crystal structure of Ulm16, along with modeling of its substrates and site-directed mutagenesis, allows for rationalization of this wide substrate scope. Overall, Ulm16 represents a promising tool for the biocatalytic production of macrocyclic peptides.

DNA‐Barcoded Plasmonic Nanostructures for Activity‐Based Protease Sensing

AbstractWe report the development of a new class of protease activity sensors called DNA‐barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide‐DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease‐mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a‐based assay as a proxy for protease activity. As proof‐of‐concept, we show activity‐based, multiplexed detection of the SARS‐CoV‐2‐associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25‐fold turn‐on signals, 100‐fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation‐dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer‐associated protease, cathepsin B, in three different patient‐derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing.

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

β-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining β-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining β-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by β-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of β-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The β-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label β-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified β-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance β-l-arabinofuranosidases in complex biological samples.

Discovery of highly efficient CRBN-recruiting HPK1-PROTAC as a potential chemical tool for investigation of scaffolding roles in TCR signaling

Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a promising target for immune-oncology therapy. It has been recently demonstrated that loss of HPK1 kinase activity can enhance T cell receptor (TCR) signaling. However, many essential functions mediated by the HPK1 scaffolding role are still beyond the reach of any kinase inhibitor. Proteolysis targeting chimera (PROTAC) has emerged as a promising strategy for pathogenic proteins degradation with the characteristics of rapid, reversible, and low-cost versus RNA interference or DNA knock-out technology. Herein we first disclosed the design, synthesis, and evaluation of a series of thalidomide-based PROTAC molecules and identified B1 as a highly efficient HPK1 degrader with DC50 value of 1.8 nM. Further mechanism investigation demonstrated that compound B1 inhibits phosphorylation of the SLP76 protein with IC50 value of 496.1 nM, and confirmed that B1 is a bona fide HPK1-PROTAC degrader. Thus, this study provides a basis for HPK1 degraders development and the candidate could be used as a potential chemical tool for further investigation of the kinase-independent signaling of HPK1 in TCR.

The Emory‐Sage‐SGC TREAT‐AD Center: Tool development for novel targets in Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases affecting over 55 million people worldwide. Over the last 20 years, AD research has focused on a limited number of potential drug targets. Given the demonstrated heterogeneity of the disease in biological and genetic components, there is a need to evaluate a broad range of therapeutic hypotheses for target prioritization and development. The Emory‐Sage‐SGC TREAT‐AD Center is generating and openly distributing validated experimental tools necessary to test target predictions generated through sequence‐based characterization of human disease state. We believe that these tools and reagents, including chemical and biological probes that target the multifaceted dysregulation in the brains of AD patients will advance the discovery of potential drug targets for AD.MethodOur Center uses integrated computational approaches to identify target predictions from a set of prioritized therapeutic hypotheses. The current target portfolio was assembled by evaluating prioritized understudied proteins by the Accelerating Medicines Partnership in AD (AMP‐AD) consortium and additional NIA‐supported AD consortia. Nominated targets were evaluated by calculating an unbiased target risk score, literature score, and druggability. Targets are then mapped to 16 biological domains (BDs) that describe and codify the different processes that are dysregulated in AD and prioritized based on multiple lines of evidence for overall AD‐risk. Targets that meet criteria for development are evaluated to identify a set of experimental reagents necessary for hypothesis testing, termed a “target enablement package (TEP).ResultWe have prioritized more than 30 understudied targets for TEP development. For each understudied target, a TEP includes expression constructs, purified protein and methods, validated knockout cell lines, and antibody validation. All reagents are developed to meet established quality criteria. For a subset of tractable targets additional TEP components include assay development, crystal structures, screening, and probe development. Advanced targets include SYK, DDX1, SFRP1, SDC4, ARHGEF2, and CAPN2. TREAT‐AD investigators place all data, knowledge, reagents, and tools into the open domain with no intellectual property claims.ConclusionAll data, protocols, reagent sets, and chemical tools will be made widely available on the AD Knowledge Portal. For more information see www.treatad.org.

Abstract C086: NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples

Abstract Two complementary mechanisms safeguard genome fidelity during DNA replication: DNA polymerase proofreading and DNA mismatch repair (MMR). DNA polymerase proofreading or MMR deficiencies can occur independently or, more rarely, simultaneously; loss of function of either or both pathways leads to the accumulation of DNA mutations. Lynch Syndrome is caused by mono-allelic germline mutations in MMR genes MLH1, MSH2, MSH6 or PMS2. Gene expression from the wild-type allele elicits sufficient MMR activity until a second hit somatic mutation inactivates the wild-type allele leading to MMR deficiency (MMR-d).  Constitutional mismatch repair deficiency (CMMRD) syndrome is an inherited early onset cancer predisposition syndrome resulting from biallelic germline mutations in MLH1, MSH2, MSH6 or PMS2. MMR-d cancers are commonly and typically characterized by accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences - microsatellite instability (MSI); importantly, such mutations occur at higher rates in genetically unstable tumour cells under replicative stress than in normal cells. Cancers that harbour >40% microsatellite variations (positive for two or more of five clinically tested microsatellite markers) are described as MSI-high (MSI-H). Importantly, MMR-d, MSI-H tumours are now eligible for immune checkpoint therapy, irrespective of tissue type, providing substantial patient benefit and a major advance in cancer treatment. This clinical context is consistent with the hypothesis that MMR-d elicits an immunogenic state. However, there is an unmet need for chemical tools to explore the role of therapeutic MMR inhibition alongside immune checkpoint blockade. Here, for the first time, we demonstrate that NP1867 is a potent, selective small molecule inhibitor of the Gyrase, Hsp90, Histidine Kinase, MutL (GHKL) family endonuclease PMS1 Homolog 2, Mismatch Repair System Component (PMS2). We demonstrate by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in cell-based assays of MMR-dependent DNA repair. NP1867 displays minimal off-target binding in broad biochemical screening panels including kinases and ATPases. Treatment of MMR-proficient, colorectal cancer cell lines with NP1867 increases tumour mutational burden and generates COSMIC mutational signatures consistent with MMR-d patient samples demonstrating, for the first time, that acute inhibition of PMS2 with a small molecule phenocopies the epigenetic silencing and/or genetic loss of MMR observed in patients. We characterise cell- and tissue-based target engagement, and in vivo pharmacokinetic and pharmacodynamic properties of NP1867 to inform optimal use by the research community for further exploring PMS2 and MMR function. Citation Format: Julian Blagg, Philippe Riou, Alexia Hervieu, Eleonora Piumatti, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Sam Weeks, Maria Rodriguez, Kalpesh Parmar, Pradip Patel, Adam Peall, Paige Tongue, Tess McLaren, Robert Workman, Ruzica Bago, Stuart Robjohns, Rebecca Beaumont, Mike Briggs, Gareth Langley, Charles Nichols, David Clark, Paul Winship, Benoit Rousseau, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C086.

Novel Pyrazinoindolones As Amyloid‐Beta Aggregation Inhibitors

AbstractBackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative condition which primarily affects the elderly population. One of hallmarks of AD is the accumulation of protein aggregates known as amyloid‐beta (Aβ) that promotes neurotoxicity and cognitive decline. Recent discoveries in the field led to the accelerated market approval of anti‐Aβ antibodies aducanumab and lecanemab. These are exciting discoveries. However, considering the cost and patient compliance issues with these biological therapies, discovering novel anti‐Aβ therapies that are based on small molecules is highly desirable due to several advantages over biological therapies including the ease of oral administration, reduced cost involved in bulk manufacturing and better stability on storage.MethodA library of novel pyrazino[1,2‐a]indole‐1(2H)‐one derivatives were designed using the computational modeling software Discovery Studio Structure‐Based‐Design (Biovia Inc, San Diego USA). Then synthetic chemistry protocols were optimized to prepare small molecule library and were characterized by 1H and 13C NMR and Liquid Chromatography Mass Spectrometry studies. The anti‐Aβ activity of these novel molecules toward Aβ40 aggregation was evaluated by fluorescence spectroscopy using the thioflavin‐T based aggregation kinetic studies. In addition, the online web tool SwissADME was used to calculate the physicochemical properties of pyrazino[1,2‐a]indole‐1(2H)‐one derivatives to assess their drug‐likeness, blood‐brain barrier permeability and pharmacokinetics.ResultA novel library of pyrazino[1,2‐a]indole‐1(2H)‐one derivatives were prepared by optimizing a four step or two step synthesis protocol starting from either phenylhydrazine or indole‐2‐carboxylic acid to afford pyrazino[1,2‐a]indole‐1(2H)‐one derivatives 5a–f. In the Aβ40 (5 μM) aggregation kinetics assay, they exhibited 14‐59% inhibition when tested at a range of concentrations (1, 5, 10 and 25 μM), demonstrating their anti‐Aβ properties. Their physicochemical properties suggest their drug‐likeness.ConclusionStructure‐activity relationship studies demonstrate that the fused tricyclic pyrazino[1,2‐a]indole‐1(2H)‐one is a suitable template to i) develop novel chemical tools to study and understand the mechanisms of Aβ40 aggregation and ii) discover novel small molecules as potential therapies for AD.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract My presentation will describe our work on the discovery, nonclinical antitumor properties, mechanism of action (MOA), identification of initial target patient population, and early clinical development of NXP-800. We discovered this agent in our academic drug discovery centre at ICR following our initial identification of the ‘bisamide’ hit CCT245232 from a diverse ~200,000-compound library using an innovative, imaging-based (ArrayScan™) phenotypic screen for inhibitors of the HSF1-mediated activation of the heat shock response in U2OS human osteosarcoma cells. Subsequent multiparameter medicinal chemistry optimisation of the bisamide series, containing the N,N′-4-methyl-1,3-phenylenediamide core, focused on establishing the cell-based SAR for both inhibition of cell proliferation and linked HSF-1 mediated HSP72 induction in the sensitive ARID1A-mutant, platinum-resistant SK-OV-3 human ovarian carcinoma cell line. This generated our orally bioavailable, in vivo-active lead compound and chemical tool CCT251236 – exhibiting oral antitumor efficacy in the SK-OV-3 human ovarian cancer xenograft model in nude mice (Cheeseman et al J Med Chem 60 180-201 2017). Final multiparameter optimization yielded the clinical development candidate CCT361814/NXP800 (Pasqua et al J Med Chem 66 5907-36 2023). With the screening hit bisamide compound already active in the single to low double digit nanomolar range, the early focus was on improving kinetic solubility while retaining the desired antiproliferative and linked HSF1 pathway inhibitory activity together with simultaneous optimisation of PK/PD properties to maximize oral antitumour efficacy; finally requiring modification to the fluorobisamide NXP800 to eliminate PGP-mediated efflux and achieve optimal PK/PD, Pharmacologic Audit Trail, antitumor efficacy and tolerability. In view of these properties, its clean safety panel profile, clear therapeutic index and acceptable dose-to-human prediction, NXP800 was progressed into clinical development. Evaluation in a mini-panel of human cancer cell lines and tumor xenografts revealed high sensitivity to NXP800 in ARIDIA-deficient human ovarian cancer models, confirmed in the large Sanger cancer cell line panel and in isogenic pair systems. Activity exceeded that of cisplatin in both platinum-sensitive and platinum-resistant ARID1A-mutant ovarian cancer xenograft models. By RNA-Seq we identified two sets of consistent, major gene expression profile changes in human cancer cell lines exposed to NXP800, namely: 1) the expected changes in HSF1-regulated genes, together with 2) activation of the integrated stress response (ISR), including ATF4-regulated gene expression. This latter did not indicate a global stress response to NXP800 as we saw no activation of the unfolded protein response (UPR). Physiologically, ISR activation is mediated by phosphorylation of EIF2α which is tightly regulated by four stress-controlled kinases, GCN2, HRI, PKR and PERK. Using each of systematic siRNA knockdown or pharmacologic inhibition by two small-molecule tool compounds from different chemotypes, we discovered that GCN2 alone was necessary and sufficient for ISR activation and cell growth inhibition by NXP800 in SK-OV-3 ovarian cancer cells. Also, orthogonal siRNA depletion or pharmacologic inhibition of GCN2 both markedly reduced the antiproliferative activity of NXP800 in these cells. Global phospho-proteome analysis demonstrated defined changes in response to NXP800 which were reversed by co-treatment with a GCN2 inhibitor. Other follow-on studies at the protein level demonstrated that pharmacological concentrations of NXP800 induced GCN2-dependent phosphorylation of EIF2α, inhibition of global cap-dependent protein translation, and selective translation of ATF4 – a transcription factor that activates downstream genes such as CHAC1 and CHOP in the ISR – both in ARID1A-mutant human ovarian cells in vitro and in corresponding tumor xenograft models in vivo. Using siRNA silencing, we showed that prevention of ATF4 induction substantially reduced the antiproliferative response of SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. Controls used in these experiments included a closely matched, inactive regioisomer. Furthermore, we showed that ISR induction by NXP800 inhibited HSF1 activation, in SK-OV-3 cells, confirming the mechanistic link between ISR activation and inhibition of HSF1-mediated transcription. We further showed that GCN2 activation was not due to the canonical mechanism of amino acid starvation. In summary, we used an HSF1 pathway-focused phenotypic screen to discover the mechanistically novel, first-in-class drug NXP800, which acts potently on ARID1A-mutant, human ovarian cancer cells to stimulate GCN2 and thereby activate the ISR pathway, leading to ATF4 induction and inhibition of HSF1-mediated gene transcription in ARID1A-mutant, platinum-resistant human ovarian cancer cells. Through this mechanism, NXP800 shows highly promising activity in models of this cancer type, including potent inhibition of proliferation in vitro and substantial regression of tumor xenografts in vivo. Studies are currently underway to determine precisely how NXP800 stimulates GCN2 activity and the role of ARID1A deficiency in its pharmacological effects. Recent ChIP-Seq studies are informing on the molecular interactions between ARID1A mutation and the expression of ISR/ATF4 and HSF1-regulated genes. With Nuvectis Pharma, the Phase 1a dose escalation study has been completed and a multicentre Phase 1b expansion cohort study in ARID1A-mutated, platinum-resistant ovarian cancer – a high unmet medical need – has been initiated (NCT05226507) in collaboration with the GOG Foundation and the European Network of Gynaecological Oncological Trial Group (ENGOT). FDA has issued a Fast Track designation to NXP800 in this setting. In addition, NXP800 shows therapeutic potential in non-clinical models of endometrial, gastro-intestinal and bile duct cancer, with FDA Orphan Drug status granted in the last of these indications. Citation Format: Paul Workman. From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA014.

Nontargeted screening method for detection of illicit adulterants in dietary supplements and herbal medicines using UHPLC-QTOF-MS with fine-tuned Spec2Vec-based spectral similarity and chemical classification filter

Liquid chromatography-mass spectrometry (LC-MS) is a widely utilized technique for inspecting adulteration. Unscrupulous businesses persistently introduce novel illegal adulterants, making it necessary to develop methods to screen compounds not present in the current library. Conventional cosine similarity for mass spectral libraries matching is limited in their ability to identify structurally similar compounds. In our previous study, comparison of performance among four advanced similarity algorithms revealed that Spec2Vec exhibited the best performance in terms of both detection capability and false discovery rate, making it the chosen method for identifying illegal adulterants. However, Spec2Vec still exhibited worse performance compared to MS2DeepScore and entropy similarity in the aspects of detection capability and false discovery rate, respectively. In this study, our objective was to optimize the performance of spectral similarity for a specific compound class by fine-tuning a pretrained Spec2Vec model. Additionally, we implemented the chemical classification tool CANOPUS to address the issue of similarities in backbone structures between illegal adulterants and compounds found in herbal medicine, which can lead to false positives. We utilized glucocorticoids as potentially illicit adulterants to provide a proof-of-concept, and the results demonstrated that the fine-tuned Spec2Vec model not only exhibits a significant improvement in detection ability compared to the original model but also achieves comparable performance to MS2Deepscore. Moreover, the fine-tuned Spec2Vec model shows notably fewer false positives in comparison to MS2Deepscore. Overall, this proposed pipeline demonstrates high effectiveness and competitiveness in inspecting illegal adulterants, enhancing the analysis of large-scale MS data.

Enhancing nicotinamide N-methyltransferase bisubstrate inhibitor activity through 7-deazaadenosine and linker modifications

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3–12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.