Research

Development of Carbamoyl Fluorides as Warheads to Target Orphan Enzymes

Code:

Z1-70004

Range:

01. March 2026 - 28. February 2029

Range:

1 FTE

Leader:

Anže Meden

Field:

1-09 Natural sciences and Mathematics - Pharmacy

Abstract:

About 10% of approved drugs form a covalent bond with their macromolecular target. Covalent drugs, previously neglected due to safety concerns, have recently taken centre stage in drug discovery programs as they pose certain advantages over non-covalent drugs, namely higher potency, longer residence time, decoupling of pharmacokinetics and pharmacodynamics, and increased selectivity. In the last three decades, activity-based protein profiling (ABPP) has emerged as an invaluable tool in chemoproteomics. Its main advantage is that it directly evaluates enzyme functionality through reaction of ABPP probe’s reactive moiety (warhead) with the enzyme’s catalytic residue, typically Cys and Ser. Serine hydrolases (SHs) represent about 1% of all human proteins and consist of serine proteases and metabolic SHs. Their common denominator is the catalytic Ser residue which is typically located within a catalytic triad (Ser–His–Asp/Glu). Approx. 40% of mammalian metabolic serine hydrolases still lack functional annotation, and virtually nothing is known about their physiological substrates and role in the organism.
One such underexplored orphan SH is ABHD14B, a lysine deacetylase that has a unique mechanism, as it transfers an acetyl group from post-translationally modified ε-acetyllysine residues of different proteins to coenzyme A. It is mostly present in metabolically active tissues, and affects the transcription of a number of genes involved in central metabolic processes – e.g., gluconeogenesis, glycolysis, Krebs cycle. Furthermore, ABHD14B is associated with a plethora of (patho)physiological processes and diseases in humans, including progression of aggressive cancers and parkinsonism. Another such orphan enzyme in bacteria is FphI from antibiotic-resistant Staphylococcus aureus which has a promising potential for diagnosis or management of MRSA infections. Very little is known about this SH besides its involvement in lipid metabolism and stress responses. For these two enzymes, no known small-molecule inhibitors or pharmacological tools are available. The development of specific, in vivo active small molecule tools is therefore of paramount importance for further studies to elucidate their function, biology, and role in various diseases, and perhaps even open innovative avenues in therapeutic approaches to these diseases.
Carbamoyl fluorides (CFs) are a class of underexplored organofluorine compounds with untapped potential for various biology- and medicinal chemistry-related uses. We have previously identified CFs as potential ABHD14B and FphI inhibitors, which was confirmed by in vitro biochemical assays and ABPP chemoproteomics. The proposed project aims to develop carbamoyl fluoride-based covalent inhibitors to characterize, detect, or modulate of these orphan SH targets through achieving 5 scientific objectives, namely: (i) to find fragment-sized CF covalent inhibitors for ABHD14B and FphI; (ii) to use structure-based design and optimization to improve potency, reactivity, selectivity, and stability of CF inhibitors; (iii) to study the catalytic mechanism of ABHD14B using enzyme kinetics, molecular dynamics, and multiscale QM/MM calculations; (iv) to identify an in vivo-applicable molecular tool for ABHD14B, and determine its in vivo selectivity profile and effects on glucose metabolism; (v) to identify a FphI-selective probe for labelling live bacteria. This will be achieved through the collaboration of scientists from four institutions – UL Faculty of Pharmacy, Indian Institute of Science Education and Research Pune, University of Otago, and University of Kentucky – who will combine their interdisciplinary expertise in a highly complementary manner. By providing the much-needed first small molecule tools for ABHD14B and FphI, biologists and medicinal chemists will be better equipped to further study these underexplored enzymes, perhaps even in context of drug design for human use.