Acylation | EPFL Graph Search

En chimie organique, une acylation est une réaction au cours de laquelle un groupement acyle est ajouté à une molécule, ce groupement étant transféré depuis un agent acylant. Les halogénures d'acyle sont très utilisés comme agents acylants. En effet, ils forment des électrophiles puissants en présence de catalyseurs de type acides de Lewis. Ainsi, l'acylation de Friedel-Crafts utilise le chlorure d'acétyle pour effectuer une acylation du benzène, en présence de chlorure d'aluminium, selon un mécanisme de type substitution électrophile aromatique. L'acylation d'un alcène permet de former une cétone, celle d'une amine permet d'obtenir un amide, et celle d'un alcool conduit à un ester. Dans ces deux derniers cas, le mécanisme de l'acylation est de type addition-élimination, l'amine ou l'alcool jouant le rôle de nucléophile. Parmi les autres agents acylants couramment utilisés, on peut également citer les anhydrides d'acide. L'acide succinique est également couramment utilisé, au cour

Methods for the generation of large combinatorial macrocycle libraries

Sevan Mleh Habeshian

Macrocycles are an attractive class of molecules due to their good binding properties and yet rather small size that allows, in many cases, crossing membranes to reach intracellular targets. In comparison to classical small molecule drugs, macrocycles are generally better at binding to challenging targets such as proteins having flat, featureless surfaces or protein-protein interactions. However, the development of macrocycle-based ligands has been challenging due to the lack of sufficiently large libraries of macrocyclic compounds for screening. The preparation of large numbers of macrocycles is limited due to the chromatographic purification that is typically required following low-yielding cyclization reactions. As a result, the full potential of macrocycles cannot be exploited in drug discovery efforts.The goal of my thesis was to develop new methods for the efficient generation of large macrocycle libraries. Towards this end, I envisioned to establish a new macrocycle library synthesis principle in which a large panel of m small cyclic peptides are combinatorially ligated in microwell plates with a large number of n chemical fragments, and the resulting m x n products screened without purification. For accessing large libraries, I planned to perform the reactions and screens at a nanomole scale and using contact-less liquid transfer.In a first project, I developed a method for obtaining readily pure macrocyclic peptides directly from solid phase. Peptide synthesis took place on a disulfide linker to solid phase, which allowed for side chain deprotection with acid without simultaneous peptide cleavage. When a thiol group was included at the N-terminus, treatment with base in DMSO resulted in an intramolecular disulfide exchange to release peptides cyclized by a disulfide bond. The method was tolerant of diverse peptide sequences, and released all tested compounds in high purity. With this method, I was able to synthesize cyclic peptides in 96-well plates, and thus hundreds of peptides in parallel with a moderate effort. I screened a test library and identified a weak inhibitor of thrombin (Ki = 13 ± 1 µM), which validated the method. In the second project, I intended to combinatorially diversify the above macrocyclic peptides with chemical fragments in nanoliter volumes, as described above. For chemical ligation, I chose to acylate amines built into the macrocyclic peptides with carboxylic acid building blocks. Following proof-of-concept experiments, I diversified a 384-member library with 10 carboxylic acids in nanoliter volumes to give 3,840 macrocyclic compounds, which I screened against thrombin. I identified a potent thrombin inhibitor (Ki = 44 ± 1 nM), and a co-crystal structure was obtained which confirmed binding to the active site of the protease. In a final application, I generated and screened a library of 19,968 macrocycles against MDM2, an important oncology target forming a protein-protein interaction with p53. The screen identified a high nanomolar binder (KD = 394 ± 41 nM) that I optimized in two rounds of iterative library synthesis and screening (KD = 31 ± 6 nM).The methods developed in the two projects are currently applied by our laboratory, and for the development of macrocycle-based ligands to diverse disease targets.

EPFL2021

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains

Muhammad Umair Anwar, Martina Audagnotto, Matteo Dal Peraro, Francisco De Magalhães Sarmento R De Mesquita, Giulia Fonti, Laurence Gouzi Abrami, Sylvia Ho, Florence Pojer, Patrick Alain Sandoz, Françoise Gisou van der Goot Grunberg

Acyl protein thioesterase APT2 interacts with membranes via its charged beta-tongue, becomes palmitoylated by ZDHHC3/7 and deforms the bilayer to extract substrate acyl chains. APT2 deacylation leads to its membrane release and degradation. Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.

NATURE RESEARCH2021

Methods for the generation of large combinatorial macrocycle libraries

Sevan Mleh Habeshian

EPFL2021