Monthly Archives: March 2024

Novel Bidentate Amine Ligand and the Interplay between Pd(II) and Pt(II) Coordination and Biological Activity

Posted on 15 March 2024 by

The novel N-[[4-(phenylmethoxy)phenyl]methyl]-2-pyridinemethanamine (L) coordinates Pt(II) and Pd(II) giving two complexes of formula [MLCl₂]. The ligand and Pd(II) complex impaired 65 % and 59 %, respectively, of SARS-CoV-2 replication at the viable concentrations of 50 μM. In the in vitro antitumor evaluation, Pt(II) complex showed significant cytotoxicity with a GI₅₀ of 10 μM, and no selectivity in the panel of tumor cells evaluated.

Abstract

Pt(II) and Pd(II) coordinating N-donor ligands have been extensively studied as anticancer agents after the success of cisplatin. In this work, a novel bidentate N-donor ligand, the N-[[4-(phenylmethoxy)phenyl]methyl]-2-pyridinemethanamine, was designed to explore the antiparasitic, antiviral and antitumor activity of its Pt(II) and Pd(II) complexes. Chemical and spectroscopic characterization confirm the formation of [MLCl₂] complexes, where M=Pt(II) and Pd(II). Single crystal X-ray diffraction confirmed a square-planar geometry for the Pd(II) complex. Spectroscopic characterization of the Pt(II) complex suggests a similar structure. ¹H NMR, ¹⁹⁵Pt NMR and HR-ESI-MS(+) analysis of DMSO solution of complexes indicated that both compounds exchange the chloride trans to the pyridine for a solvent molecule with different reaction rates. The ligand and the two complexes were tested for in vitro antitumoral, antileishmanial, and antiviral activity. The Pt(II) complex resulted in a GI₅₀ of 10.5 μM against the NCI/ADR-RES (multidrug-resistant ovarian carcinoma) cell line. The ligand and the Pd(II) complex showed good anti-SARS-CoV-2 activity with around 65 % reduction in viral replication at a concentration of 50 μM.

Biocatalytic Regioselective O‐acylation of Sesquiterpene Lactones from Chicory: A Pathway to Novel Ester Derivatives

Posted on 15 March 2024 by

This article reports the first biocatalytic modification of sesquiterpene lactones (STL) found in chicory plants. Regioselective O-acylation of their primary alcohol group is achieved by the immobilized lipase B from Candida antarctica, with various aliphatic vinyl esters as acyl donors. High conversion rates were observed, facilitating the synthesis of novel semi-synthetic STL ester derivatives with promising pharmaceutical applications.

Abstract

We report the first biocatalytic modification of sesquiterpene lactones (STLs) found in the chicory plants, specifically lactucin (Lc), 11β,13-dihydrolactucin (DHLc), lactucopicrin (Lp), and 11β,13-dihydrolactucopicrin (DHLp). The selective O-acylation of their primary alcohol group was carried out by the lipase B from Candida antarctica (CAL-B) using various aliphatic vinyl esters as acyl donors. Perillyl alcohol, a simpler monoterpenoid, served as a model to set up the desired O-acetylation reaction by comparing the use of acetic acid and vinyl acetate as acyl donors. Similar conditions were then applied to DHLc, where five novel ester chains were selectively introduced onto the primary alcohol group, with conversions going from >99 % (acetate and propionate) to 69 % (octanoate). The synthesis of the corresponding O-acetyl esters of Lc, Lp, and DHLp was also successfully achieved with near-quantitative conversion. Molecular docking simulations were then performed to elucidate the preferred enzyme-substrate binding modes in the acylation reactions with STLs, as well as to understand their interactions with crucial amino acid residues at the active site. Our methodology enables the selective O-acylation of the primary alcohol group in four different STLs, offering possibilities for synthesizing novel derivatives with significant potential applications in pharmaceuticals or as biocontrol agents.

13Cβ‐Valine and 13Cγ‐Leucine Methine Labeling To Probe Protein Ligand Interaction

Posted on 15 March 2024 by

A novel labeling approach introduces isolated ¹³C−H spin systems to the methyl-deuterated side chains of leucine and valine residues. Ligands with known binding geometries have been analyzed in combination with corresponding protein samples, showing that chemical shift perturbation data correlate well with the distance- and angle parameters of CH-π hydrogen bonds.

Abstract

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed ¹H chemical shift perturbation resulting from CH-π interaction of H_β-Val and H_γ-Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

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.

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.

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.

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.

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.

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.

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.