Category Archives: ChemBioChem

Light‐Activatable Photocaged UNC2025 for Triggering TAM Kinase Inhibition in Bladder Cancer

Posted on 18 March 2024 by

Photopharmacology: three photoactivatable small molecules based on the UNC2025 scaffold were developed for controlling TAM kinase activity in vitro and in cellulo assays. The dicaged compound 3 proved to be the most striking with a complete loss of inhibition while recovering full activity upon light irradiation.

Abstract

Photopharmacology is an emerging field that utilizes photo-responsive molecules to enable control over the activity of a drug using light. The aim is to limit the therapeutic action of a drug at the level of diseased tissues and organs. Considering the well-known implications of protein kinases in cancer and the therapeutic issues associated with protein kinase inhibitors, the photopharmacology is seen as an innovative and alternative solution with great potential in oncology. In this context, we developed the first photocaged TAM kinase inhibitors based on UNC2025, a first-in-class small molecule kinase inhibitor. These prodrugs showed good stability in biologically relevant buffer and rapid photorelease of the photoremovable protecting group upon UV-light irradiation (<10 min.). These light-activatable prodrugs led to a 16-fold decrease to a complete loss of kinase inhibition, depending on the protein and the position at which the coumarin-type phototrigger was introduced. The most promising candidate was the N,O-dicaged compound, showing the superiority of having two photolabile protecting groups on UNC2025 for being entirely inactive on TAM kinases. Under UV-light irradiation, the N,O-dicaged compound recovered its inhibitory potency in enzymatic assays and displayed excellent antiproliferative activity in RT112 cell lines.

Endogenous Production and Vibrational Analysis of Heavy‐Isotope‐Labeled Peptides from Cyanobacteria

Posted on 15 March 2024 by

Microcystis aeruginosa cultures were grown in ¹⁵N- and ¹³C-enriched medium to produce heavy-labeled cyanopeptides, which were characterized with vibrational spectroscopy and mass spectrometry. Up to 98 % heavy isotope incorporation was observed for the peptides, which displayed strong vibrational bands associated with diene, thiazole, and phenyl functional groups.

Abstract

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in ¹⁵N/¹³C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC–MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Systematic Analysis of the MIO‐forming Residues of Aromatic Ammonia Lyases

Posted on 15 March 2024 by nYannik Brack, nChenghai Sun, nDong Yi, nUwe T. Bornscheuern

Through a systematic analysis of protein sequences, several protein clusters of AAL-like enzymes with unusual MIO-forming motifs (ACG, TSG, SSG, and CSG) were identified, including two novel HALs with CSG motifs, one PAM with a TSG motif, and three novel ETLs without MIO motif. The systematic study and mutagenesis of MIO-groups in this work enable thus functional prediction and mutagenesis research of AALs.

Abstract

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA

Posted on 15 March 2024 by nJagandeep S. Saraya, nDerek K. O'Flahertyn

Tandem Oligonucleotide Synthesis (TOS) of (chemically modified) DNA and RNA using a commercially available cleavable linker. We show that the 2,2′-sulfonyldiethylene linker is effectively incorporated in DNA/RNA sequences, and efficiently cleaved during standard nucleic acid deprotection protocols. Moreover, we utilize our TOS approach to perform ligation experiments to incorporate N3′→P5′ phosphoramidate, and pyrophosphate linkages in DNA strands

Abstract

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2′-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.

Photo‐affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids

Posted on 15 March 2024 by

Leon Duque, Vallavoju, and colleagues report the design and development of photo-affinity and metabolic labeling probes based on the opioid alkaloids. These chemical probes are used to measure localization and protein interactions in a cellular context by confocal microscopy and chemical proteomics. These insights will inform the development of future probes inspired by the opioid alkaloids.

Abstract

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

Chemo‐Enzymatic Derivatization of Glycerol‐Based Oligomers: Structural Elucidation and Potential Applications

Posted on 15 March 2024 by

Fatty acids- and peptide-functionalized glycerol-based oligoesters synthesized in this work and their potential applications based on the performed derivatization.

Abstract

Switching from oil-based to bio-based feedstocks to ensure the green transition to a sustainable and circular future is one of the most pressing challenges faced by many industries worldwide. For the cosmetics and personal and house care industries there is a strong drive to accelerate this transition from the customers that starts favoring the purchase of naturally derived and bio-degradable products over the traditionally available products. In this work we developed a series of fully biobased macromolecules constituted of a glycerol-based oligoester backbone. Based on the subsequent derivatization with fatty acids or peptides, the resulting products may find application as emulsifiers, wetting agents, and potential vectors for the delivery of bioactive peptides. All steps of the resulting macromolecules were conducted following the green chemistry principles with no toxic or environmentally damaging compounds that were used in the overall production process.

Tripartite Split‐GFP for High Throughput Screening of Small Molecules: A Powerful Strategy for Targeting Transient/Labile Interactors like E2‐E3 Ubiquitination Enzymes

Posted on 15 March 2024 by nCécile Polge, nStéphanie Cabantous, nDaniel Taillandiern

Use of the in cellulo tripartite split-GFP assay for High Throughput Screening (HTS) of small molecules inhibiting an E2-E3 Protein-Protein Interaction (PPI). E3 and E2 are fused to GFP10 and GFP11 tags. When an E3 and an E2 interact, GFP10 and GFP11 come close together and reconstitute functional GFP. Molecules preventing the E2-E3 interaction prevent the reconstitution of a functional GFP

Abstract

The search for inhibitors of the Ubiquitin Proteasome System (UPS) is an expanding area, due to the crucial role of UPS enzymes in several diseases. The complexity of the UPS and the multiple protein-protein interactions (PPIs) involved, either between UPS proteins themselves or between UPS components and theirs targets, offer an incredibly wide field for the development of chemical compounds for specifically modulating or inhibiting metabolic pathways. However, numerous UPS PPIs are transient/labile, due the processivity of the system (Ubiquitin [Ub] chain elongation, Ub transfer, etc.). Among the different strategies that can be used either for deciphering UPS PPI or for identifying/characterizing small compounds inhibitors, the split-GFP approach offers several advantages notably for high throughput screening of drugs. Split-GFP is based on the principle of protein-fragment complementation assay (PCA). PCA allows addressing PPIs by coupling each protein of interest (POI) to fragments of a reporter protein whose reconstitution is linked to the interaction of the POI. Here, we review the evolution of the split-GFP approach from bipartite to tripartite Split-GFP and its recent applicability for screening chemical compounds targeting the UPS.

Molybdenum‐Copper Antagonism In Metalloenzymes And Anti‐Copper Therapy

Posted on 15 March 2024 by nBiplab K. Maiti, nIsabel Moura, nJosé J. G. Mouran

This review highlights the key lessons learned on Mo−Cu interaction in Mo/Cu-CODH and ORP and how it controls the elevated cellular Cu level involved in WD and cancer through Cu−TTM therapy.

Abstract

The connection between 3d (Cu) and 4d (Mo) via the “Mo−S−Cu” unit is called Mo−Cu antagonism. Biology offers case studies of such interactions in metalloproteins such as Mo/Cu−CO Dehydrogenases (Mo/Cu−CODH), and Mo/Cu Orange Protein (Mo/Cu−ORP). The CODH significantly maintains the CO level in the atmosphere below the toxic level by converting it to non–toxic CO₂ for respiring organisms. Several models were synthesized to understand the structure–function relationship of these native enzymes. However, this interaction was first observed in ruminants, and they convert molybdate (MoO₄ ²⁻) into tetrathiomolybdate (MoS₄ ²⁻; TTM), reacting with cellular Cu to yield biological unavailable Mo/S/Cu cluster, then developing Cu-deficiency diseases. These findings inspire the use of TTM as a Cu-sequester drug, especially for treating Cu-dependent human diseases such as Wilson diseases (WD) and cancer. It is well known that a balanced Cu homeostasis is essential for a wide range of biological processes, but negative consequence leads to cell toxicity. Therefore, this review aims to connect the Mo−Cu antagonism in metalloproteins and anti-copper therapy.

Preclinical Evaluation of Azabenzimidazole‐Based PET Radioligands for γ‐8 Dependent Transmembrane AMPA Receptor Regulatory Protein Imaging

Posted on 15 March 2024 by

A novel azabenzimidazoles-based¹⁸F-labeled positron emission tomography (PET) ligand ([¹⁸F]TARP-2205) is synthesized and preliminarily evaluated. In vitro autoradiography studies indicate that these PET ligands possess high in vitro specific binding to TARP γ-8.

Abstract

AMPA glutamate receptors (AMPARs) play a pivotal role in excitatory neurotransmission, particularly in the hippocampus where the TARP γ-8 subunit is enriched and serves as a target for emerging anti-epileptic drugs. To enable in vivo visualization of TARP γ-8 distribution and expression by positron emission tomography (PET), this study focuses on the development of novel ¹⁸F-labeled TARP γ-8 inhibitors and their corresponding precursors, stemming from the azabenzimidazole scaffold. The resulting radioligands [¹⁸F]TARP-2204 and [¹⁸F]TARP-2205 were successfully synthesized with acceptable radiochemical yield, high molar activity, and excellent radiochemical purity. In vitro autoradiography demonstrates high level of specific binding of [¹⁸F]TARP-2205 to TARP γ-8 in both rat and nonhuman primate brain tissues. However, unexpected radiodefluorination in PET imaging studies of rodents emphasizes the need for further structural refinement. This work serves as an excellent starting point for the development of future ¹⁸F-labeled TARP γ-8 PET tracers, offering valuable insights into medicinal chemistry design, radiosynthesis and subsequent PET evaluation.

Investigating Protein Binding with the Isothermal Ligand‐induced Resolubilization Assay

Posted on 15 March 2024 by

Isothermal Ligand-Induced Resolubilization Assay (ILIRA) is a new screening method for identifying protein ligands that relies on differences in solubility between unliganded and liganded proteins under lyotropic stress conditions. In this proof-of-concept study, we demonstrate the application of ILIRA to different protein targets and present alternative assay modalities.

Abstract

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.