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The mammalian membrane‐bound O‐acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small‐molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small‐molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure‐activity relationship investigation identified single enantiomer IMP‐1575, the most potent HHAT inhibitor reported to‐date, and guided design of photocrosslinking probes that maintained HHAT‐inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small‐molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP‐1575 (K i=38 nM) and a strategy for mapping small molecule interaction sites in MBOATs., Structure‐activity relationship analysis of Hedgehog acyltransferase (HHAT) inhibitors allowed rational design of photochemical probes for HHAT. Probe photocrosslinking identified the first small‐molecule inhibitor binding site in HHAT and revealed the inhibitory mechanism, providing an optimal HHAT tool inhibitor IMP‐1575 (K i=38 nM) for future studies.

Kallikrein‐related peptidase 14 (KLK14) is one of the several secreted KLK serine proteases involved in prostate cancer (PCa) pathogenesis. While relatively understudied, recent reports have identified KLK14 as overexpressed during PCa development. However, the modulation of KLK14 expression during PCa progression and the molecular and biological functions of this protease in the prostate tumor microenvironment remain unknown. To determine the modulation of KLK14 expression during PCa progression, we analyzed the expression levels of KLK14 in patient samples using publicly available databases and immunohistochemistry. In order to delineate the molecular mechanisms involving KLK14 in PCa progression, we integrated proteomic, transcriptomic, and in vitro assays with the goal to identify substrates, related‐signaling pathways, and functional roles of this protease. We showed that KLK14 expression is elevated in advanced PCa, and particularly in metastasis. Additionally, KLK14 levels were found to be decreased in PCa tissues from patients responsive to neoadjuvant therapy compared to untreated patients. Furthermore, we also identified that KLK14 expression reoccurred in patients who developed castrate‐resistant PCa. The combination of proteomic and transcriptomic analysis as well as functional assays revealed several new KLK14 substrates (agrin, desmoglein 2, vitronectin, laminins) and KLK14‐regulated genes (Interleukin 32, midkine, SRY‐Box 9), particularly an involvement of the mitogen‐activated protein kinase 1 and interleukin 1 receptor pathways, and an involvement of KLK14 in the regulation of cellular migration, supporting its involvement in aggressive features of PCa progression. In conclusion, our work showed that KLK14 expression is associated with the development of aggressive PCa suggesting that targeting this protease could offer a novel route to limit the progression of prostate tumors. Additional work is necessary to determine the benefits and implications of targeting/cotargeting KLK14 in PCa as well as to determine the potential use of KLK14 expression as a predictor of PCa aggressiveness or response to treatment., Kallikrein‐related peptidase 14 (KLK14) is a secreted serine protease belonging to the kallikrein‐related‐peptidase family. We identified that KLK14 is overexpressed in advanced prostate cancer and acts on various substrates involved in adhesion, migration, and invasion of cancer cells, thus modulating genes and signaling pathways essential for prostate cancer progression. These results suggest that KLK14 is a potential target to control aggressiveness of prostate tumors.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

Protein myristoylation plays key roles in biological processes, for instance, in membrane attachment and activation of proteins and in mediating protein-protein and protein-lipid interactions. Furthermore, myristoylated proteins are involved in disorders, including cancer and viral infections. Therefore, new tools to study protein myristoylation are in high demand. Herein, we report the development of photoactivatable probes, based on a diazirine-substituted analogue of myristic acid. The probes bind to and, upon irradiation, covalently label the lipid-binding chaperone protein uncoordinated 119 (UNC119). UNC119 increases overall solubility and regulates specifically the transport of myristoylated proteins between intercellular membranes. The binding mode of the probes is similar to that of the myristate moiety, and the residues inside the hydrophobic pocket of UNC119 proteins that are critical for covalent binding have been identified. The interaction with UNC119 was also demonstrated in cell lysate by means of affinity enrichment. Moreover, it is shown that the myristate analogue can be incorporated into peptide substrates by N-myristoyl transferases of Leishmania and Trypanosoma protozoan parasites.

Cysteine -­ reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high -­ quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan -­ reactive compounds. Here we describe quantitative irreversible tethering (qIT) , a general method for screening cysteine -­ reactive small molecules based upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and g uide further optimization. The power of this technique is highlighted by the identification of a Cdk2 -­ selective allosteric (type IV) kinase inhibitor whose novel mode -­ of -­ action could be exploited therapeutically.

Peptide aptamers are powerful chemical genetic tools for the dissection of biological networks, but their application to in vivo systems has been limited by the challenging problem of targeting peptides to a specific site on a single target protein. Here we present our initial research on a novel technique for targeting combinatorial peptide aptamers to a protein binding-site using a small-molecule binding-partner (or ‘Trojan horse’). Novel peptide-based inhibitors for Plasmodium falciparum myristoyl-CoA:protein N-myristoyl transferase (PfNMT) have been selected from a one-bead one-compound library using a high-throughput on-bead screening methodology, targeted to the active site of NMT with a myristate (C14 : 0 fatty acid) substrate analogue. From an initial screen of an unbiased 130321-compound library of lipid/combinatorial peptide chimeras, we have selected 6-mer peptides in an on-bead assay which show NMT inhibition with IC50 values ranging down to low micromolar.

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.

Low temperature Lewis acid catalysed oxygen to carbon rearrangements of anomerically linked 6-substituted tetrahydropyranyl enol ethers lead to the corresponding trans-ketones in a highly diastereoselective manner, whereas at higher temperatures the cis-ketones are formed with a high degree of selectivity under thermodynamic control.

A range of enol ethers, linked via their oxygen atom to the anomeric centre of a pyran ring system, was shown to undergo oxygen to carbon rearrangement upon treatment with a Lewis acid to give the corresponding 2-carbon substituted products. At low temperature, trimethylsilyl trifluoromethanesulfonate catalysed rearrangements of anomerically linked 6-substituted tetrahydropyranyl enol ethers gave selectively the trans-pyranyl ketones, whereas at higher temperatures selective formation of the cis-pyranyl ketones was observed. In a simple application of the methodology the cis-selective rearrangement was used as the key step in a concise total synthesis of a constituent of civet.