Database-driven in silico-identification and characterization of novel aldoxime dehydratases.

Aldoxime dehydratases (Oxds) are a unique class of enzymes, which catalyzes the dehydration of aldoximes to nitriles in an aqueous environment. Recently, they gained attention as a catalyst for a green and cyanide-free alternative to established nitrile syntheses, which often require the use of toxic cyanides and harsh reaction conditions. Up to now only thirteen aldoxime dehydratases have been discovered and biochemically characterized. This raised the interest for identifying further Oxds with, e.g., complementary properties in terms of substrate scope. In this study, 16 novel genes, presumably encoding aldoxime dehydratases, were selected by using a commercially available 3DM database based on OxdB, an Oxd from Bacillus sp. OxB-1. Out of 16 proteins, six enzymes with aldoxime dehydratases activity were identified, which differ in their substrate scope and activity. While some novel Oxds showed better performance for aliphatic substrate such as n-octanaloxime compared to the well characterized OxdRE from Rhodococcus sp. N-771, some showed activity for aromatic aldoximes, leading to an overall high usability of these enzymes in organic chemistry. The applicability for organic synthesis was underlined by converting 100 mM n-octanaloxime at a 10 mL scale within 5 h with the novel aldoxime dehydratase OxdHR as whole-cell catalyst (33 mgbww/mL).

Alternative use of BRAF inhibitors in patients with metastatic melanoma unable to swallow pills.

INTRODUCTION: BRAF and MEK inhibitors have been approved for use in metastatic melanoma therapies. All of them are administered as oral capsules or pills. We report two cases treated applying an alternative method of vemurafenib or debrafenib-trametinib administration in patients unable to swallow. CASE REPORT: The first case involved a 38-year-old man who was referred to a dermatologist for dysphagia and anorexia. After a computerized tomography (CT) scan it was concluded that the dysphagia was due to compression by mediastinal metastasis in a context of metastatic BRAF mutant melanoma. The second case involved a 35-year-old man who was diagnosed in March 2017 with melanoma of the back of the hand. Several months later a positron emission tomography (PET)/CT scan was performed. It revealed multiple disseminated metastasis.Management & Outcome: The first patient presented total dysphagia and was unable to swallow pills. It was decided to dissolve vemurafenib in order to facilitate administration. Dysphagia was improved 48 hours later, and oral feeding was reintroduced. Due to severe tablet phobia, the second patient was unable to swallow pills. Dabrafenib capsules were emptied and trametinib pills were grinded. One month later, we noted improved health associated with reduction of the metastases. DISCUSSION: Our study highlights the possibility of crushing or dissolving BRAF and MEK inhibitors in metastatic melanoma patients for whom it is impossible to swallow pills, eliciting a response and achieving significant if temporary clinical benefit.

Significance of tumor protein p53 mutation in cellular process and drug selection in brain lower grade (WHO grades II and III) glioma.

Aim: Tumor protein p53 (TP53) mutant is one of the most frequently mutated genes in glioma. Results: The Cancer Genome Atlas data has shown that TP53 mutation is present in 49% of lower grade (World Health Organization [WHO] grades II and III) glioma patients. Data from The Genomics of Drug Sensitivity in Cancer database showed that three drugs: (5Z)-7-oxozeaenol, dabrafenib and nutlin-3a (-), have shown more resistance in patients with TP53 mutation. We identified 1100 differentially expressed genes. Functional enrichment analysis showed that the differentially expressed genes are mainly concentrated in the transport of ionic and cancer-related pathways. The top ten hub genes were identified and an outcome analysis revealed the most critical genes related to prognosis. Conclusion: Our results identified the key genes and pathways that might provide the basic proof to improve individualized treatment in patients with glioma.

Discovery of orally active indirubin-3'-oxime derivatives as potent type 1 FLT3 inhibitors for acute myeloid leukemia.

FMS-like receptor tyrosine kinase-3 (FLT3) is expressed on acute leukemia cells and is implicated in the survival, proliferation and differentiation of hematopoietic cells in most acute myeloid leukemia (AML) patients. Despite recent achievements in the development of FLT3-targeted small-molecule drugs, there are still unmet medical needs related to kinase selectivity and the progression of some mutant forms of FLT3. Herein, we describe the discovery of novel orally available type 1 FLT3 inhibitors from structure-activity relationship (SAR) studies for the optimization of indirubin derivatives with biological and pharmacokinetic profiles as potential therapeutic agents for AML. The SAR exploration provided important structural insights into the key substituents for potent inhibitory activities of FLT3 and in MV4-11 cells. The profile of the most optimized inhibitor (36) showed IC50 values of 0.87 and 0.32 nM against FLT3 and FLT3/D835Y, respectively, along with potent inhibition against MV4-11 and FLT3/D835Y expressed MOLM14 cells with a GI50 value of 1.0 and 1.87 nM, respectively. With the high oral bioavailability of 42.6%, compound 36 displayed significant in vivo antitumor activity by oral administration of 20 mg/kg once daily dosing schedule for 21 days in a mouse xenograft model. The molecular docking study of 36 in the homology model of the DFG-in conformation of FLT3 resulted in a reasonable binding mode in type 1 kinases similar to the reported type 1 FLT3 inhibitors Crenolanib and Gilteritinib.

CITCO Directly Binds to and Activates Human Pregnane X Receptor.

The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are activated by structurally diverse chemicals to regulate the expression of target genes, and they have overlapping regulation in terms of ligands and target genes. Receptor-selective agonists are, therefore, critical for studying the overlapping function of PXR and CAR. An early effort identified 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) as a selective human CAR (hCAR) agonist, and this has since been widely used to distinguish the function of hCAR from that of human PXR (hPXR). The selectivity was demonstrated in a green monkey kidney cell line, CV-1, in which CITCO displayed >100-fold selectivity for hCAR over hPXR. However, whether the selectivity observed in CV-1 cells also represented CITCO activity in liver cell models was not hitherto investigated. In this study, we showed that CITCO: 1) binds directly to hPXR; 2) activates hPXR in HepG2 cells, with activation being blocked by an hPXR-specific antagonist, SPA70; 3) does not activate mouse PXR; 4) depends on tryptophan-299 to activate hPXR; 5) recruits steroid receptor coactivator 1 to hPXR; 6) activates hPXR in HepaRG cell lines even when hCAR is knocked out; and 7) activates hPXR in primary human hepatocytes. Together, these data indicate that CITCO binds directly to the hPXR ligand-binding domain to activate hPXR. As CITCO has been widely used, its confirmation as a dual agonist for hCAR and hPXR is important for appropriately interpreting existing data and designing future experiments to understand the regulation of hPXR and hCAR. SIGNIFICANCE STATEMENT: The results of this study demonstrate that 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is a dual agonist for human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). As CITCO has been widely used to activate hCAR, and hPXR and hCAR have distinct and overlapping biological functions, these results highlight the value of receptor-selective agonists and the importance of appropriately interpreting data in the context of receptor selectivity of such agonists.

Biological Evaluation of Noscapine analogues as Potent and Microtubule-Targeted Anticancer Agents.

In present investigation, an attempt was undertaken to modify the C-9 position of noscapine (Nos), an opium alkaloid to yield 9 -hydroxy methyl and 9 -carbaldehyde oxime analogues for augmenting anticancer potential. The synthesis of 9-hydroxy methyl analogue of Nos was carried out by Blanc reaction and 9-carbaldehyde oxime was engineered by oxime formation method and characterized using FT-IR, 1H NMR, 13C NMR, mass spectroscopy, and so on techniques. In silico docking techniques informed that 9-hydroxy methyl and 9-carbaldehyde oxime analogues of Nos had higher binding energy score as compared to Nos. The IC50 of Nos was estimated to be 46.8 µM signficantly (P < 0.05) higher than 8.2 µM of 9-carbaldehyde oxime and 4.6 µM of 9-hydroxy methyl analogue of Nos in U87, human glioblastoma cells. Moreover, there was significant (P < 0.05) difference between the IC50 of 9-carbaldehyde oxime and 9-hydroxy methyl analogue of Nos. Consistent to in vitro cytotoxicity data, 9-hydroxy methyl analogue of Nos induced significantly (P < 0.05) higher degree of apoptosis of 84.6% in U87 cells as compared to 78.5% and 64.3% demonstrated by 9-carbaldehyde oxime and Nos, respectively. Thus the higher therapeutic efficacy of 9-hydroxy methyl analogue of Nos may be credited to higher solubility and inhibitory constant (K).

Inhibition Mechanisms of Indoleamine 2,3-Dioxygenase 1 (IDO1).

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the rate-limiting step in the kynurenine pathway of tryptophan metabolism, which is involved in immunity, neuronal function, and aging. Its implication in pathologies such as cancer and neurodegenerative diseases has stimulated the development of IDO1 inhibitors. However, negative phase III clinical trial results of the IDO1 inhibitor epacadostat in cancer immunotherapy call for a better understanding of the role and the mechanisms of IDO1 inhibition. In this work, we investigate the molecular inhibition mechanisms of four known IDO1 inhibitors and of two quinones in detail, using different experimental and computational approaches. We also determine for the first time the X-ray structure of the highly efficient 1,2,3-triazole inhibitor MMG-0358. Based on our results and a comprehensive literature overview, we propose a classification scheme for IDO1 inhibitors according to their inhibition mechanism, which will be useful for further developments in the field.

Labeling of VEGFR1D2 through oxime ligation.

We reported an useful protocol for the labeling of the second domain of the Vascular Endothelial Growth Factor Receptor 1 (VEGFR1D2), a small protein ligand able to bind VEGF, the main regulator of angiogenesis. We developed a bioconjugation strategy based on the use of oxime-ligation reaction conjugating an aldehyde derivative of the VEGFR1D2 to a molecular probe harboring an alkoxyamine functional group. We applied the synthetic protocol to prepare a biotinylated conjugate of VEGFR1D2 and we demonstrate that the bioconjugate retains its ability to specifically bind its natural ligand, VEGF, with high affinity. The biotinylated VEGFR1D2 could be useful to detect and quantify VEGF for diagnostic purposes as well as a tool for the screening of new molecules targeting VEGFRs for therapeutic applications. The labeling protocol is versatile and can be extended to different molecular probes, such as fluorophores, chelators or multimeric scaffolds, affording a biomedical platform for VEGF targeting.

Biochemical Characterization of Full-Length Oncogenic BRAFV600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases.

The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full-length BRAFV600E is available. Herein, we show the successful purification of active, full-length BRAFV600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell-based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. Firstly, steady-state kinetic analyses reveal that purified BRAFV600E is more active than fully activated wild-type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAFV600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side-to-side dimer interface. Thirdly, BRAFV600E bypasses inhibitory P-loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAFV600E , complementing the understanding of BRAF regulation under normal and disease conditions.

Resistance mechanism of the oncogenic ß3-αC deletion mutation in BRAF kinase to dabrafenib and vemurafenib revealed by molecular dynamics simulations and binding free energy calculations.

BRAF kinase is an essential target for anti-cancer drug development. Emergence of the ß3-αC loop deletion mutation (ΔNVTAP) in BRAF kinase frequently occurred in human cancers seriously compromises the therapeutic efficacy of some BRAF kinase inhibitors, such as dabrafenib and vemurafenib. However, the mechanism of this resistance is still not well understood. In this study, the influence of the ß3-αC deletion mutation on the binding profiles of three BRAF kinase inhibitors (AZ628, dabrafenib, and vemurafenib) with BRAFV600E or BRAFΔNVTAP was explored by conventional molecular dynamics (MD) simulations and binding free energy calculations. The simulation results indicated that the ß3-αC deletion mutation enhances the flexibility of the αC helix and alters their conformations, which amplify the conformational entropy change (-TΔS) and weaken the interactions between the inhibitors and BRAF. The further per-residue binding free energy decomposition analysis revealed that the ΔNVTAP mutation changed the contributions of a few key residues to the bindings of dabrafenib or vemurafenib, such as L57, L66, W83, C84, F135, G145, and F147, but did not have obvious impact on the contributions of these residues to AZ628. Our results provide valuable clues to understand the mechanisms of drug resistance conferred by the ß3-αC deletion mutation.

Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit Improved Physicochemical Properties and Efficient Reactivation of Human Acetylcholinesterase Inhibited by Several Nerve Agents.

Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the p Ka value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to nonchlorinated analogues. The nucleophilicity was examined and the p Ka was found to be lower than that of analogous nonchlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the dichlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was, respectively, 3-, 7-, and 8-fold higher than by K027. Its universality, PAMPA permeability, favorable acid dissociation constant coupled with its negligible cytotoxic effect, and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an antidote for organophosphorus poisoning.

Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non-invasive pharmacological therapies. Here, we present the results of several unbiased small-molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology-based target prediction tool to integrate the results with the whole-transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small-molecule-based therapies. We found indirubin-3-monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss-of-CCM phenotypes in human endothelial cells. Our multi-organism-based approach reveals new components of the CCM regulatory network and foreshadows novel small-molecule-based therapeutic applications for suppressing this devastating disease in patients.

PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in phosphonocystoximic acid biosynthesis in Streptomyces.

Several oxime-containing small molecules have useful properties, including antimicrobial, insecticidal, anticancer, and immunosuppressive activities. Phosphonocystoximate and its hydroxylated congener, hydroxyphosphonocystoximate, are recently discovered oxime-containing natural products produced by Streptomyces sp. NRRL S-481 and Streptomyces regensis NRRL WC-3744, respectively. The biosynthetic pathways for these two compounds are proposed to diverge at an early step in which 2-aminoethylphosphonate (2AEPn) is converted to (S)-1-hydroxy-2-aminoethylphosphonate ((S)-1H2AEPn) in S. regensis but not in Streptomyces sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or (S)-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from Streptomyces sp. NRRL S-481 and S. regensis NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual N-oxidases. Here, we show that recombinant PcxL and HpxL catalyze the FAD- and NADPH-dependent oxidation of 2AEPn and 1H2AEPn, producing a mixture of the respective aldoximes and nitrosylated phosphonic acid products. Measurements of catalytic efficiency indicated that PcxL has almost an equal preference for 2AEPn and (R)-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than (R)-1H2AEPn under saturating conditions, resulting in a similar but slightly lower kcat/Km We observed that (S)-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in S. regensis. HpxL also used both 2AEPn and (R)-1H2AEPn, with the latter inhibiting HpxL at high concentrations. Bioinformatic analysis indicated that PcxL and HpxL are members of a new class of oxime-forming N-oxidases that are broadly dispersed among bacteria.

Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA.

Herein we report the first exploration of a dual-targeting drug design strategy to improve the efficacy of small-molecule cancer immunotherapy. New hybrids of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors and DNA alkylating nitrogen mustards that respectively target IDO1 and DNA were rationally designed. As the first-in-class examples of such molecules, they were found to exhibit significantly enhanced anticancer activity in vitro and in vivo with low toxicity. This proof-of-concept study has established a critical step toward the development of a novel and effective immunotherapy for the treatment of cancers.

5-Bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime: A long-acting anticancer agent and a suicide inhibitor for epoxide hydrolase.

Indirubin 3'-oxime (Indox (1b)) suppresses cancer cell growth (IC50: 15µM towards HepG2 cells) and inhibits cell cycle-related kinases such as cyclin-dependent kinases and glycogen synthase kinase-3ß. We have previously reported that the conjugation of 1b with oxirane, a protein-reactive component, enhanced the cytotoxic activity of Indox as determined from the IC50 value (1.7µM) of indirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/Ind (1c)). Here we prepared Epox/Ind derivatives with one or two halogen atoms or a methoxy group on the aromatic ring(s) of an Indox moiety and studied the structure-activity relationships of the substituent(s). We found that bromine-substitution at the 5-position on 1c or any Epox/Ind derivative(s) having bromine on the aromatic ring except Epox/6'-Br-Ind was efficient to improving anticancer activity. Of the 22 Epox/Ind derivatives, 5-bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/5-Br-Ind (2c)) was the best anticancer agent in both short- (24h) (IC50: 0.67µM) and extended-duration (72h) cultures. The high anticancer activity of 2c was partly due to it being a poor substrate and a suicide inhibitor for epoxide hydrolase as epoxide hydrolase was identified as the enzyme primarily responsible for the metabolism of 2c.

The Binding Mode of N-Hydroxyamidines to Indoleamine 2,3-Dioxygenase 1 (IDO1).

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important target in cancer immunotherapy. The most advanced clinical compound, epacadostat (INCB024360), binds to the heme cofactor of IDO1 through an N-hydroxyamidine function. Conflicting binding modes have recently been proposed, reporting iron binding either through the hydroxyamidine oxygen or through the hydroxyamidine nitrogen atom. Here, we use quantum chemical calculations, docking, and quantum mechanics/molecular mechanics calculations based on available X-ray data to resolve this issue and to propose a physically meaningful binding mode. Our findings will aid the design of novel IDO1 ligands based on this pharmacophore.

Simple and cost-effective liquid chromatography-mass spectrometry method to measure dabrafenib quantitatively and six metabolites semi-quantitatively in human plasma.

Dabrafenib is an inhibitor of BRAF V600E used for treating metastatic melanoma but a majority of patients experience adverse effects. Methods to measure the levels of dabrafenib and major metabolites during treatment are needed to allow development of individualized dosing strategies to reduce the burden of such adverse events. In this study, an LC-MS/MS method capable of measuring dabrafenib quantitatively and six metabolites semi-quantitatively is presented. The method is fully validated with regard to dabrafenib in human plasma in the range 5-5000 ng/mL. The analytes were separated on a C18 column after protein precipitation and detected in positive electrospray ionization mode using a Xevo TQ triple quadrupole mass spectrometer. As no commercial reference standards are available, the calibration curve of dabrafenib was used for semi-quantification of dabrafenib metabolites. Compared to earlier methods the presented method represents a simpler and more cost-effective approach suitable for clinical studies. Graphical abstract Combined multi reaction monitoring transitions of dabrafenib and metabolites in a typical case sample.

In Vitro Interactions of Epacadostat and its Major Metabolites with Human Efflux and Uptake Transporters: Implications for Pharmacokinetics and Drug Interactions.

Epacadostat (EPAC) is a first-in-class, orally active inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 and has demonstrated promising clinical activity. In humans, three major plasma metabolites have been identified: M9 (a glucuronide-conjugate), M11 (a gut microbiota metabolite), and M12 (a secondary metabolite formed from M11). It is proposed, based on the human pharmacokinetics of EPAC, that the biliary excretion of M9, the most abundant metabolite, leads to the enterohepatic circulation of EPAC. Using various in vitro systems, we evaluated in the present study the vitro interactions of EPAC and its major metabolites with major drug transporters involved in drug absorption and disposition. EPAC is a substrate for efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), but it is not a substrate for hepatic uptake transporters [organic anion transporting polypeptides OATP1B1 and OATP1B3]. The low permeability of M9 suggests an essential role for transporters in its disposition. M9 is likely excreted from hepatocytes into bile via multidrug resistance-associated protein 2 (MRP2) and BCRP, excreted into blood via MRP3, and transported from blood back into hepatocytes via OATP1B1 and OATP1B3. M11 and M12 are not substrates for P-gp, OATP1B1 or OATP1B3, and M11, but not M12, is a substrate for BCRP. With respect to inhibition of drug transporters, the potential of EPAC, M9, M11, and M12 to cause clinical drug-drug interactions via inhibition of P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, or organic cation transporter 2 was estimated to be low. The current investigation underlines the importance of metabolite-transporter interactions in the disposition of clinically relevant metabolites, which may have implications for the pharmacokinetics and drug interactions of parent drugs.

Prediction of the Transporter-Mediated Drug-Drug Interaction Potential of Dabrafenib and Its Major Circulating Metabolites.

The BRAF inhibitor dabrafenib was recently approved for the treatment of certain BRAF V600 mutation-positive tumors, either alone or in combination therapy with the mitogen-activated extracellular signal regulated kinase 1 (MEK1) and MEK2 inhibitor, trametinib. This article presents the dabrafenib transporter-mediated drug-drug interaction (DDI) risk assessment, which is currently an important part of drug development, regulatory submission, and drug registration. Dabrafenib and its major circulating metabolites (hydroxy-, carboxy-, and desmethyl-dabrafenib) were investigated as inhibitors of the clinically relevant transporters P-gp, BCRP, OATP1B1, OATP1B3, OCT2, OAT1, and OAT3. The DDI Guidance risk assessment decision criteria for inhibition of BCRP, OATP1B1 and OAT3 were slightly exceeded and therefore a minor DDI effect resulting from inhibition of these transporters remained possible. Biliary secretion is the major excretion pathway of dabrafenib-related material (71.1% of orally administered radiolabeled dose recovered in feces), whereas urinary excretion was observed as well (22.7% of the dose). In vitro uptake into human hepatocytes of the dabrafenib metabolites, but not of dabrafenib parent compound, was mediated, at least in part, by hepatic uptake transporters. The transporters responsible for uptake of the pharmacologically active hydroxy- and desmethyl dabrafenib could not be identified, whereas carboxy-dabrafenib was a substrate of several OATPs. Dabrafenib, hydroxy-, and desmethyl-dabrafenib were substrates of P-gp and BCRP, whereas carboxy-dabrafenib was not. Although a small increase in exposure to carboxy-dabrafenib upon inhibition of OATPs and an increase in exposure to desmethyl-dabrafenib upon inhibition of P-gp or BCRP cannot be excluded, the clinical significance of such increases is likely to be low.

Application of oxime-diversification to optimize ligand interactions within a cryptic pocket of the polo-like kinase 1 polo-box domain.

By a process involving initial screening of a set of 87 aldehydes using an oxime ligation-based strategy, we were able to achieve a several-fold affinity enhancement over one of the most potent previously known polo-like kinase 1 (Plk1) polo-box domain (PBD) binding inhibitors. This improved binding may result by accessing a newly identified auxiliary region proximal to a key hydrophobic cryptic pocket on the surface of the protein. Our findings could have general applicability to the design of PBD-binding antagonists.