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RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation., Competing Interests: Declaration of interests Authors on this manuscript are employed by Promega. Promega owns patents related to target engagement using BRET., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer., (© 2022. The Author(s).)

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 ( 1a ), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 ( 1s ), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 ( 1a ). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 ( 1s ). Importantly, we demonstrate that SW-101 ( 1s ) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN 2. Taken together, these results bode well for the further development of SW-101 ( 1s ) as a disease-modifying HDAC6i.

Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi's) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi's and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi's and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi's, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability to improve upon memory performance in a mouse model of FXS, Fmr1 -/- mice. This small molecule demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC 50 = 2.3 nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms. Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the impaired acetylated α-tubulin levels in the hippocampus of Fmr1 -/- mice. Lastly, SW-100 ameliorates several memory and learning impairments in Fmr1 -/- mice, thus modeling the intellectual deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based therapies for the treatment of this rare disease.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Positive effects have been observed in offspring from beef cows supplemented with corn dried distillers grain with solubles (DDGS) during late gestation. The hypothesis of this study was that late gestational DDGS supplementation to beef cows would increase blood flow (BF) to the gravid uterus and mammary gland thus impacting birthweight and post-natal growth of the offspring. Experiment 1 investigated mammary gland BF in multiparous cows during late pregnancy. Beef cows were fed a control (CON1) diet of low-quality hay (n = 5) or a supplement diet (SUP1) of low-quality hay with DDGS [1.7 g/kg of body weight (BW); n = 6]. In Experiment 2, multiparous late pregnant beef cows were fed either a control (CON2) diet of a low-quality hay (n = 4) or a supplement diet (SUP2) of low-quality hay with DDGS (1.7 g/kg of BW; n = 5). Uterine and mammary gland BF were recorded every 21 days during late gestation. In Experiment 1, there were no effects of diet or day on mammary gland hemodynamics. In Experiment 2, total and ipsilateral uterine BF was less (p ≤ 0.04) in SUP2 vs. CON2 cows and similar BF to contralateral horns. Mammary gland BF was unaltered by maternal supplementation. Even when measured in two different years in two different environments, mammary gland BF remained unaltered to DDGS supplementation. Investigations on the mechanism that may impact uterine BF during late gestation remain to be known., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC 50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC 50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

Background: Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity., New Method: In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long-term., Results: We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration., Comparison With Existing Methods: Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact., Conclusions: These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders., (Copyright © 2014. Published by Elsevier B.V.)

Chromobacterium violaceum was recovered at necropsy from the lungs, liver, spleen, and an interscapular abscess of a Chinese red panda (strain 98-9187) [J. Vet. Diagn. Invest. 12 (2000) 177]. As the lungs exhibited extensive, necrotizing lesions harboring bacterial aggregates, we sought to determine whether C. violaceum produced an elastase that might in part account for these lesions. The C. violaceum type strain (ATCC 12472(T)) and strain 98-9187 were shown to exhibit elastolytic activity by elastin Congo red and elastin nutrient agar assays. The activity was isolated from the periplasmic fraction and was present throughout the growth cycle. Activity increased markedly in late logarithmic phase growth. In elastin-limiting medium, activity rapidly decreased in early stationary phase indicating a tight regulation of yield. The activity was optimal at neutral pH and was sensitive to the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. Activity was restored upon addition of zinc indicating the enzyme is a zinc metalloproteinase. A band corresponding to purified elastase activity was present at approximately 30kDa in a denaturing polyacrylamide gel.

Pseudomonas aeruginosa LasB elastase gene (lasB) transcription is controlled by the two-component quorum-sensing system of LasR, and the autoinducer, 3OC(12)-HSL (N-3-[oxododecanoyl]homoserine lactone). LasR and 3OC(12)-HSL-mediated lasB activation requires a functional operator sequence (OP1) in the lasB promoter region. Optimal activation of lasB, however, requires a second sequence of 70% identity to OP1, named OP2, located 43 bp upstream of OP1. In this study, we used sequence substitutions and insertion mutations in lasBp-lacZ fusion plasmids to explore the role of OP2 in lasB activation. Our results demonstrate that (i) OP1 and OP2 synergistically mediate lasB activation; (ii) OP2, like OP1, responds to LasR and 3OC(12)-HSL; and (iii) the putative autoinducer-binding domain of LasR is not required for synergistic activation from OP1 and OP2.