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.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase.

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/ß-catenin signalling, Hippo signalling and telomere maintenance. The ARCs recognise a conserved tankyrase-binding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrase's poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent "scaffolding" mechanisms contribute to tankyrase function. Here we report a fragment-based screening programme against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR) spectroscopy. We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions.

Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species.

CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies.

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.

Pyrido[3,4-d]pyrimidin-4(3H)-one metabolism mediated by aldehyde oxidase is blocked by C2-substitution.

1. We have previously described C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one derivatives as cell permeable inhibitors of the KDM4 and KDM5 subfamilies of JmjC histone lysine demethylases. 2. Although exemplar compound 1 exhibited moderate clearance in mouse liver microsomes, it was highly cleared in vivo due to metabolism by aldehyde oxidase (AO). Similar human and mouse AO-mediated metabolism was observed with the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold and other C8-substituted derivatives. 3. We identified the C2-position as the oxidation site by LC-MS and 1H-NMR and showed that C2-substituted derivatives are no longer AO substrates. 4. In addition to the experimental data, these observations are supported by molecular modelling studies in the human AO protein crystal structure.

A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms.

The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.

Synthesis and Evaluation of a 2,11-Cembranoid-Inspired Library.

The 2,11-cembranoid family of natural products has been used as inspiration for the synthesis of a structurally simplified, functionally diverse library of octahydroisobenzofuran-based compounds designed to augment a typical medicinal chemistry library screen. Ring-closing metathesis, lactonisation and SmI2 -mediated methods were exemplified and applied to the installation of a third ring to mimic the nine-membered ring of the 2,11-cembranoids. The library was assessed for aqueous solubility and permeability, with a chemical-space analysis performed for comparison to the family of cembranoid natural products and a sample set of a screening library. Preliminary investigations in cancer cells showed that the simpler scaffolds could recapitulate the reported anti-migratory activity of the natural products.

Structure enabled design of BAZ2-ICR, a chemical probe targeting the bromodomains of BAZ2A and BAZ2B.

The bromodomain containing proteins BAZ2A/B play essential roles in chromatin remodeling and regulation of noncoding RNAs. We present the structure based discovery of a potent, selective, and cell active inhibitor 13 (BAZ2-ICR) of the BAZ2A/B bromodomains through rapid optimization of a weakly potent starting point. A key feature of the presented inhibitors is an intramolecular aromatic stacking interaction that efficiently occupies the shallow bromodomain pockets. 13 represents an excellent chemical probe for functional studies of the BAZ2 bromodomains in vitro and in vivo.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Phytochemical and antifungal studies on Terminalia mollis and Terminalia brachystemma.

Phytochemical investigation of the stem bark of Terminalia mollis afforded friedelin (1), catechin with epicatechin (2), gallocatechin with epigallocatechin (3) and 3-O-methylellagic acid 4'-O-alpha-rhamnopyranoside (4). Arjunolic acid with 2alpha, 3beta, 23-trihydroxy-urs-12-en-28-oic acid (5), 2alpha-hydroxyursolic acid (6), gallic acid (7), chebulanin (8) and 2''-O-galloylvitexin (9) were isolated from the leaf. Chebulanin (8), betulinic acid (10), ursolic acid (11), catechin (12), isoorientin (13), orientin (14), isovitexin (15) and punicalagin (16) were isolated from Terminalia brachystemma leaf. The first full unambiguous NMR assignments for (4) and (8), and revised assignments for (9), are reported. Compound (16) showed good activity against three Candida species.

Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeasts.

Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdilution method by including a colorimetric indicator-resazurin for end-point determination. This modified method showed good reproducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some traditional uses of these plants while assisting the identification of the active ingredients.