Monitoring how, when, and where small molecules engage their targets inside living cells is a critical step in chemical biology and pharmacological research, because it enables compound efficacy and confirmation of mode of action to be assessed. In this mini-review we summarize the currently available methodologies to detect and prove direct target engagement in cells and offer a critical view of their key advantages and disadvantages. As the interest of the field shifts toward discovery and validation of high-quality agents, we expect that efforts to develop and refine these types of methodologies will also intensify in the near future.

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (K <10 nm), interference with Ras signaling and growth inhibition require 5-20 μm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.

The sucCD gene of Advenella mimigardefordensis strain DPN7 T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ⌬sucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD Am ) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD Am is Mg 2؉ or Mn 2؉ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V max ‫؍‬ 9.85 ؎ 0.14 mol min ؊1 mg ؊1 , K m ‫؍‬ 0.143 ؎ 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V max ‫؍‬ 0.12 ؎ 0.01 mol min ؊1 mg ؊1 ) and the affinity was 6-fold lower (K m ‫؍‬ 0.818 ؎ 0.046 mM). Based on the present results, we conclude that SucCD Am is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.3,3Ј-Dithiodipropionic acid (DTDP) is an organic disulfide and a precursor for the production of polythioesters (PTEs) by bacteria (25). Further applications for DTDP are thermodynamic studies (40), development of secondary batteries (52), amino acid analysis (53), and the construction of self-assembling monolayers (10). Microbial production of PTEs from simple carbon sources and inorganic sulfur is currently not possible. Knowledge of the catabolism of organic sulfur compounds in bacteria could provide a reasonable strategy to engineer strains suitable for PTE production. A first step in this direction was the isolation of bacteria able to utilize DTDP as the sole source of carbon and energy. Advenella mimigardefordensis strain DPN7T , a betaproteobacterium, found in mature compost in a waste management facility was one of the isolates (15,56

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6d impairs KRas spatial organization and signaling in cells.H owever, despite potent binding in vitro (K D < 10 nm), interference with Ras signaling and growth inhibition require 5-20 mm compound concentrations.W edemonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6d by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6d with up to 7h ydrogen bonds,r esulting in picomolar affinity.T heir release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6d interaction may impair the growth of tumors driven by oncogenic KRas.

No abstract

Woodward's reagent K (WRK) is a reactive heterocyclic compound that has been employed in protein chemistry to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. We have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.

In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16∆pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating other CoA-transferase(s) or CoA-synthetase(s), thereby compensating for the lacking Pct. The ability of R. eutropha H16 to substitute absent enzymes by isoenzymes has been already shown in different other studies in the past.

Background: Hitherto, bacterial thiol dioxygenases were only rarely identified and characterized, and the catabolism of mercaptosuccinate is scarcely known. Results: Mercaptosuccinate dioxygenase catalyzes exclusively the conversion of mercaptosuccinate yielding succinate and sulfite. Conclusion: Mercaptosuccinate dioxygenase represents a novel thiol dioxygenase and is the key enzyme during mercaptosuccinate degradation. Significance: The identification of this thiol dioxygenase provides new insights in substrate specificity of cupin motif containing dioxygenases.