Determination of Ligand-Binding Affinity (Kd) Using Transverse Relaxation Rate (R2) in the Ligand-Observed 1H NMR Experiment and Applications to Fragment-Based Drug Discovery.

High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low µM to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.

Evaluation of APOBEC3B Recognition Motifs by NMR Reveals Preferred Substrates.

APOBEC3B (A3B) deamination activity on ssDNA is considered a contributing factor to tumor heterogeneity and drug resistance in a number of human cancers. Despite its clinical impact, little is known about A3B ssDNA substrate preference. We have used nuclear magnetic resonance to monitor the catalytic turnover of A3B substrates in real-time. This study reports preferred nucleotide sequences for A3B substrates, including optimized 4-mer oligonucleotides, and reveals a breadth of substrate recognition that includes DNA sequences known to be mutated in drug-resistant cancer clones. Our results are consistent with available clinical and structural data and may inform the design of substrate-based A3B inhibitors.

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone.

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(-/-) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(-/-) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(-/-) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation.

Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α.

BACKGROUND: Endoplasmic reticulum stress, caused by the presence of misfolded proteins, activates the stress sensor inositol-requiring enzyme 1α (IRE1α). The resulting increase in IRE1α RNase activity causes sequence-specific cleavage of X-box binding protein 1 (XBP1) mRNA, resulting in upregulation of the unfolded protein response and cellular adaptation to stress. The precise mechanism of human IRE1α activation is currently unclear. The role of IRE1α kinase activity is disputed, as results from the generation of various kinase-inactivating mutations in either yeast or human cells are discordant. Kinase activity can also be made redundant by small molecules which bind the ATP binding site. We set out to uncover a role for IRE1α kinase activity using wild-type cytosolic protein constructs. RESULTS: We show that concentration-dependent oligomerisation is sufficient to cause IRE1α cytosolic domain RNase activity in vitro. We demonstrate a role for the kinase activity by showing that autophosphorylation enhances RNase activity. Inclusion of the IRE1α linker domain in protein constructs allows hyperphosphorylation and further enhancement of RNase activity, highlighting the importance of kinase activity. We show that IRE1α phosphorylation status correlates with an increased propensity to form oligomeric complexes and that forced dimerisation causes great enhancement in RNase activity. In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed. CONCLUSIONS: Taken together these experiments support the hypothesis that phosphorylation is important in modulating IRE1α RNase activity which is achieved by increasing the propensity of IRE1α to dimerise. This work supports the development of IRE1α kinase inhibitors for use in the treatment of secretory cancers.

Fragment-based screening maps inhibitor interactions in the ATP-binding site of checkpoint kinase 2.

Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors.

Identification of autophosphorylation inhibitors of the inositol-requiring enzyme 1 alpha (IRE1α) by high-throughput screening using a DELFIA assay.

Inositol-requiring enzyme 1 alpha (IRE1α) is a transmembrane sensor protein with both kinase and ribonuclease activity, which plays a crucial role in the unfolded protein response (UPR). Protein misfolding in the endoplasmic reticulum (ER) lumen triggers dimerization and subsequent trans-autophosphorylation of IRE1α. This leads to the activation of its endoribonuclease (RNase) domain and splicing of the mRNA of the transcriptional activator XBP1, ultimately generating an active XBP1 (XBP1s) implicated in multiple myeloma survival. Previously, we have identified human IRE1α as a target for the development of kinase inhibitors that could modulate the UPR in human cells, which has particular relevance for multiple myeloma and other secretory malignancies. Here we describe the development and validation of a 384-well high-throughput screening assay using DELFIA technology that is specific for IRE1α autophosphorylation. Using this format, a focused library of 2312 potential kinase inhibitors was screened, and several novel IRE1α kinase inhibitor scaffolds were identified that could potentially be developed toward new therapies to treat multiple myeloma.

Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSßR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 µM, and CDK9-cyclinT with IC(50)=0.11 µM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 µM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.

Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by (32)P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.

Known drug space as a metric in exploring the boundaries of drug-like chemical space.

In this work, marketed drug compounds (or known drug space) were used as a metric to test the principles of eliminating parent structures of the nitrenium ion (aryl-amine/nitro compounds) as well as sulphur and halogen containing molecules from screening compound collections. Molecules containing such moieties and/or atoms have biological and physiochemical properties, which possibly make them less attractive as leads in drug development. It was found that precursors to the nitrenium ion were relatively abundant in known drug space at 14%. Thus, their simple elimination from drug-like chemical space is not advisable. Interestingly, the mutagenic potential of the nitrenium ions is linked to their stability and quantum mechanical calculations can be used to estimate it. Furthermore, 24% of drugs investigated contained sulphur atoms and around 28% were halogenated. As some sulphur containing moieties were abundant whilst others were scarce, it was deduced that it would be more effective to eliminate specific molecular scaffolds rather than all sulphur containing molecules. In conclusion, it has been shown that by statistically analysing known drug space a better understanding of the boundaries of drug-like chemical space was established which can help medicinal chemists in finding rewarding regions of chemical space.

Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate.

An evaluation of the ability of pifithrin-alpha and -beta to inhibit p53 function in two wild-type p53 human tumor cell lines.

The small-molecule compound pifithrin-alpha (PFT-alpha) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-alpha is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-beta. Both compounds showed limited solubility with PFT-alpha precipitating out of tissue culture medium at concentrations >30 micromol/L. PFT-alpha and -beta exhibited cytotoxic effects in vitro towards two human wild-type p53-expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC(50) values for both cell lines were 21.3 +/- 8.1 micromol/L for PFT-alpha and 90.3 +/- 15.5 micromol/L for PFT-beta, mean +/- SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-alpha enhanced cytotoxicity, particularly at higher concentrations of PFT-alpha. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation-induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-alpha nor -beta can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.

Synthesis, characterization, and 32p-postlabeling analysis of DNA adducts derived from the environmental contaminant 3-nitrobenzanthrone.

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. 3-NBA forms DNA adducts in rodent tissues that arise principally through reduction to N-hydroxy-3-aminobenzanthrone (N-OH-ABA), esterification to its acetate or sulfate ester, and reaction of this activated ester with DNA. We detected 3-NBA-derived DNA adducts in rodent tissues by (32)P-postlabeling and generated them chemically by acid-catalyzed reaction of N-OH-ABA with DNA, but their structural identification has not yet been reported. We have now prepared 3-NBA-derived adducts by reaction of a possible reactive metabolite, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA), with purine nucleosides and nucleotides, characterized them, and have shown that they are present in DNA treated with this 3-NBA derivative. Three of these adducts have been characterized as the C-C adduct N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone, the C-N adduct N-acetyl-N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone, and an unusual 3-acetylaminobenzanthrone adduct of deoxyadenosine, which involves a double linkage between adenine and benzanthrone (N1 to C1, N(6) to C11b), creating a five-membered imidazo type ring system. According to IUPAC fused ring conventions, we propose the following systematic name for this adduct: (9'-(2' '-deoxyribofuranosyl))purino[6',1':2,3]imidazo[5,4-p](1,11b-dihydro-(N-acetyl-3-amino))benzanthrone. The 3'-phosphates of these novel adducts could be 5'-postlabeled using [gamma-(32)P]ATP, although the efficiency of labeling was found to be low (less than 20%). However, none of these adducts could be detected in DNA from 3-NBA-treated rats by (32)P-postlabeling. Two of these synthetic adducts were treated with alkali to generate nonacetylated adducts, and these were also shown by HPLC to differ from those adducts found in rat DNA. Therefore, a different approach to the synthesis of authentic standards is needed for the structural characterization of 3-NBA-derived DNA adducts formed in vivo.