Category Archives: ChemBioChem

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity

Posted on 10 October 2023 by

The generation of CRL4^CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs) is currently still a largely empirical process and the design principles are still to be deciphered. Herein, a design strategy to generate CRL4^CRBN PROTACs selective for a target of interest by dialling-out the molecular glue function of thalidomide derivatives is reported.

Abstract

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4^CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4^CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4^CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4^CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

Bispidine as a Versatile Scaffold: From Topological Hosts to Transmembrane Transporters

Posted on 10 October 2023 by

We delineate a highly versatile strategy for the synthesis of topologically intriguing molecule and self-assembling macrocycles. The self-assembly of the macrocycle to nanotube displays unconventional interactions such as dihydrogen bonding. The nanotubular assembly acts as a channel for transmembrane ion transport as evident from patch clamp experiments.

Abstract

The development of designer topological structures is a synthetically challenging endeavor. We present herein bispidine as a platform for the design of molecules with various topologies and functions. The bispidine-based acyclic molecule, which shows intriguing S-shape topology, is discussed. Single-crystal X-ray diffraction studies revealed that this molecule exists in the solid state as two conformational enantiomers. In addition, bispidine-based designer macrocycles were synthesized and investigated for ionophoric properties. Patch clamp experiments revealed that these macrocycles transport both anions and cations non-specifically with at least tenfold higher chloride conductance over the cations under the given experimental conditions. Ultramicroscopy and single-crystal X-ray crystallographic studies indicated that the self-assembling macrocycle forms a tubular assembly. Our design highlights the use of unconventional dihydrogen interactions in nanotube fabrication.

Biotransformation Of l‐Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440

Posted on 10 October 2023 by

Arcyriaflavin A was produced in Pseudomonas putida KT2440 using genes from Lentzea aerocolonigenes. Cultivation conditions and supply of the precursor l-tryptophan were optimized. Genetic engineering of outer membrane vesicle release and cultivation with polyurethane adsorbent resulted in product enrichment in the supernatant. Overall, 4.7 mg arcyriaflavin A were obtained from 1 L bacterial culture representing a ten-fold increase in productivity.

Abstract

Natural products such as indolocarbazoles are a valuable source of highly bioactive compounds with numerous potential applications in the pharmaceutical industry. Arcyriaflavin A, isolated from marine invertebrates and slime molds, is one representative of this group and acts as a cyclin D1-cyclin-dependent kinase 4 inhibitor. To date, access to this compound has mostly relied on multi-step total synthesis. In this study, biosynthetic access to arcyriaflavin A was explored using recombinant Pseudomonas putida KT2440 based on a previously generated producer strain. We used a Design of Experiment approach to analyze four key parameters, which led to the optimization of the bioprocess. By engineering the formation of outer membrane vesicles and using an adsorbent in the culture broth, we succeeded to increase the yield of arcyriaflavin A in the cell-free supernatant, resulting in a nearly eight-fold increase in the overall production titers. Finally, we managed to scale up the bioprocess leading to a final yield of 4.7 mg arcyriaflavin A product isolated from 1 L of bacterial culture. Thus, this study showcases an integrative approach to improve biotransformation and moreover also provides starting points for further optimization of indolocarbazole production in P. putida.

Biotransamination of Furan‐Based Aldehydes with Isopropylamine: Enzyme Screening and pH Influence

Posted on 10 October 2023 by

The biotransamination of furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF) with isopropylamine has been studied at different substrate and amine donor concentrations with a series of amine transaminases. These processes have been thoroughly optimized taking into account several parameters, being the pH a key factor due to its influence on the in situ formation of undesired (poly)imines.

Abstract

Furan-based amines are highly valuable compounds which can be directly obtained via reductive amination from easily accessible furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). Herein the biocatalytic amination of these carbonyl derivatives is disclosed using amine transaminases (ATAs) and isopropylamine (IPA) as amine donors. Among the different biocatalysts tested, the ones from Chromobacterium violaceum (Cv-TA), Arthrobacter citreus (ArS-TA), and variants from Arthrobacter sp. (ArRmut11-TA) and Vibrio fluvialis (Vf-mut-TA), afforded high levels of product formation (>80 %) at 100–200 mM aldehyde concentration. The transformations were studied in terms of enzyme and IPA loading. The pH influence was found as a key factor and attributed to the imine/aldehyde equilibrium that can arise from the high reactivity of the carbonyl substrates with a nucleophilic amine such as IPA.

Biotransformation of Polyunsaturated Fatty Acids to Trioxilins by Lipoxygenase from Pleurotus sajor‐caju

Posted on 10 October 2023 by

Usually, polyunsaturated fatty acids (PUFAs) are converted to trioxylins (TrXs) by lipoxygenase (LOX) along with epoxide hydrolase. Here, LOX from Pleurotus sajor-caju directly converts PUFAs to TrXs with catalytic activities of hydroxylation, epoxidation, and hydrolysis of the epoxy group as an efficient TrXs-producing enzyme.

Abstract

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 μmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB₃, 13,14,15-TrXB₄, and 15,16,17-TrXB₅, respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB₃ (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB₄ (79 %), and 7.2 mM of 15,16,17-TrXB₅ (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.

Ligand‐Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Posted on 10 October 2023 by

We report ligand-directed chemistry for covalent proximity labelling of two model β-glucosidases employing small molecule iminosugar based probes in a proof of concept study. Successful enzyme labelling with respective designed and synthesized probes was determined by fluorescent readout of SDS page. Catalytic enzyme activity was maintained after the labelling process.

Abstract

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.

A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1‐epi‐Cubenol from Nonomuraea coxensis

Posted on 10 October 2023 by nJeroen S. Dickschat, nZhiyang Quan, nGregor Schnakenburgn

A high yielding sesquiterpene synthase for (+)-1-epi-cubenol from the actinomycete Nonomuraea coxensis was investigated for its cyclisation mechanism by isotopic labelling experiments. Several derivatives of (+)-1-epi-cubenol and of the plant derived compounds (−)-cubenol and (−)-1-epi-cubenol have been prepared, including an epoxide that is known as a natural product from brown algae.

Abstract

A terpene synthase from Nonomuraea coxensis was identified as (+)-1-epi-cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations. Epoxidations of (+)-1-epi-cubenol and of the plant derived compounds (−)-cubenol and (−)-1-epi-cubenol confirmed the structure of a natural product isolated from the brown alga Dictyopteris divaricata and allowed to conclude on its absolute configuration. The crystal structures of the epoxides from (+)- and (−)-1-epi-cubenol and the acid catalysed conversion into an isomeric ketone are reported.

Development of Multivalent Conjugates with a Single Non‐Canonical Amino Acid

Posted on 10 October 2023 by

Development of a sequential preparation of multivalent bioconjugates using a single non-canonical amino acid. Through the genetic incorporation of a halogenated alkynyl amino acid a 1,3-dipolar cycloadditon followed by a Sonogashira cross-coupling can facilitate the introduction of two unique moieties to a protein in a site-specific fashion.

Abstract

Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.

Towards Ruthenium(II)‐Rhenium(I) Binuclear Complexes as Photosensitizers for Photodynamic Therapy

Posted on 9 October 2023 by

The preparation of a bimetallic complex, which combines the known photophysical properties of a rhenium(I) tricarbonyl complex and a ruthenium(II) polypyridyl complex and that could act as a PS for PDT, is described.

Abstract

The search for new metal-based photosensitizers (PSs) for anticancer photodynamic therapy (PDT) is a fast-developing field of research. Knowing that polymetallic complexes bear a high potential as PDT PSs, in this study, we aimed at combining the known photophysical properties of a rhenium(I) tricarbonyl complex and a ruthenium(II) polypyridyl complex to prepare a ruthenium-rhenium binuclear complex that could act as a PS for anticancer PDT. Herein, we present the synthesis and characterization of such a system and discuss its stability in aqueous solution. In addition, one of our complexes prepared, which localized in mitochondria, was found to have some degree of selectivity towards two types of cancerous cells: human lung carcinoma A549 and human colon colorectal adenocarcinoma HT29, with interesting photo-index (PI) values of 135.1 and 256.4, respectively, compared to noncancerous retinal pigment epithelium RPE1 cells (22.4).

Tuning the Solution Self‐Assembly of a Peptide‐PEG (Polyethylene Glycol) Conjugate with α‐Cyclodextrin

Posted on 9 October 2023 by

The influence of alpha-cyclodextrin (αCD) on the aqueous solution self-assembly of peptide-polymer conjugate YYKLVFF-PEG3K is examined. Cryo-TEM and SAXS show that the conjugate self-assembles into β-sheet fibrils in aqueous solution, but complexation with αCD leads to free-floating nanosheets in aqueous solution (with a β-sheet structure). The transition from fibrils to nanosheets is driven by an increase in the number of αCD molecules threaded on the PEG chains, as determined by ¹H NMR spectroscopy.

Abstract

Cyclodextrins are saccharide ring molecules which act as host cavities that can encapsulate small guest molecules or thread polymer chains. We investigate the influence of alpha-cyclodextrin (αCD) on the aqueous solution self-assembly of a peptide-polymer conjugate YYKLVFF-PEG3K previously studied by our group [Castelletto et al., Polym. Chem., 2010, 1, 453–459]. This conjugate comprises a designed amyloid-forming peptide YYKLVFF that contains the KLVFF sequence from Amyloid β peptide, Aβ16-20, along with two aromatic tyrosine residues to enhance hydrophobicity, as well as polyethylene glycol PEG with molar mass 3 kg mol⁻¹. The conjugate self-assembles into β-sheet fibrils in aqueous solution. Here we show that complexation with αCD instead generates free-floating nanosheets in aqueous solution (with a β-sheet structure). The nanosheets comprise a bilayer with a hydrophobic peptide core and highly swollen PEG outer layers. The transition from fibrils to nanosheets is driven by an increase in the number of αCD molecules threaded on the PEG chains, as determined by ¹H NMR spectroscopy. These findings point to the use of cyclodextrin additives as a powerful means to tune the solution self-assembly in peptide-polymer conjugates and potentially other polymer/biomolecular hybrids.