Exploiting Vector Pattern Diversity of Molecular Scaffolds for Cheminformatics Tasks in Drug Discovery.

Chemical diversity is challenging to describe objectively. Despite this, various notions of chemical diversity are used throughout the medicinal chemistry optimization process in drug discovery. In this work, we show the usefulness of considering exploited vectors during different phases of the drug design process to provide a quantitative and objective description of chemical diversity. We have developed a concise and fast approach to enumerate and analyze the exploited vector patterns (EVPs) of molecular compound series, which can then be used in archetypal compound selection tasks, from hit matter identification to hit expansion and lead optimization. We first show that EVPs can be used to assess the progressibility of compounds in a fragment library design exercise. By considering EVPs, we then show how a set of compounds can be prioritized for hit expansion using EVP-based, customizable diversity sampling approaches, reducing the time taken and mitigating human biases. We also show that EVPs are a useful tool to analyze SAR data, offering the chance to uncover correlations between different vectors without predetermining the molecular scaffold structures. The codes used to perform these tasks are presented as easy-to-use Jupyter notebooks, which can be readily adapted for further related tasks.

Applications of Systematic Molecular Scaffold Enumeration to Enrich Structure-Activity Relationship Information.

Establishing structure-activity relationships (SARs) in hit identification during early stage drug discovery is important in accelerating hit confirmation and expansion. We describe the development of EnCore, a systematic molecular scaffold enumeration protocol using single atom mutations, to enhance the application of objective scaffold definitions and to enrich SAR information from analysis of high-throughput screening output. A list of 43 literature medicinal chemistry compound series, each containing a minimum of 100 compounds, published in the Journal of Medicinal Chemistry was collated to validate the protocol. Analysis using the top representative Level 1 scaffolds this list of literature compound series demonstrated that EnCore could mimic the scaffold exploration conducted when establishing SAR. When EnCore was applied to analyze an HTS library containing over 200 000 compounds, we observed that over 70% of the molecular scaffolds matched extant scaffolds within the library after enumeration. In particular, over 60% of the singleton scaffolds with only one representative compound were found to have structurally related compounds after enumeration. These results illustrate the potential of EnCore to enrich SAR information. A case study using literature cyclooxygenase-2 inhibitors further demonstrates the advantage of EnCore application in establishing SAR from structurally related scaffolds. EnCore complements literature enumeration methods in enabling changes to the physicochemical properties of molecular scaffolds and structural modifications to aliphatic rings and linkers. The enumerated scaffold clusters generated would constitute a comprehensive collection of scaffolds for scaffold morphing and hopping.

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.

Increasing the coverage of medicinal chemistry-relevant space in commercial fragments screening.

Analyzing the chemical space coverage in commercial fragment screening collections revealed the overlap between bioactive medicinal chemistry substructures and rule-of-three compliant fragments is only ∼25%. We recommend including these fragments in fragment screening libraries to maximize confidence in discovering hit matter within known bioactive chemical space, while incorporation of nonoverlapping substructures could offer novel hits in screening libraries. Using principal component analysis, polar and three-dimensional substructures display a higher-than-average enrichment of bioactive compounds, indicating increasing representation of these substructures may be beneficial in fragment screening.

Locating sweet spots for screening hits and evaluating pan-assay interference filters from the performance analysis of two lead-like libraries.

The efficiency of automated compound screening is heavily influenced by the design and the quality of the screening libraries used. We recently reported on the assembly of one diverse and one target-focused lead-like screening library. Using data from 15 enzyme-based screenings conducted using these libraries, their performance was investigated. Both libraries delivered screening hits across a range of targets, with the hits distributed across the entire chemical space represented by both libraries. On closer inspection, however, hit distribution was uneven across the chemical space, with enrichments observed in octants characterized by compounds at the higher end of the molecular weight and lipophilicity spectrum for lead-like compounds, while polar and sp(3)-carbon atom rich compounds were underrepresented among the screening hits. Based on these observations, we propose that screening libraries should not be evenly distributed in lead-like chemical space but be enriched in polar, aliphatic compounds. In conjunction with variable concentration screening, this could lead to more balanced hit rates across the chemical space and screening hits of higher ligand efficiency will be captured. Apart from chemical diversity, both screening libraries were shown to be clean from any pan-assay interference (PAINS) behavior. Even though some compounds were flagged to contain PAINS structural motifs, some of these motifs were demonstrated to be less problematic than previously suggested. To maximize the diversity of the chemical space sampled in a screening campaign, we therefore consider it justifiable to retain compounds containing PAINS structural motifs that were apparently clean in this analysis when assembling screening libraries.

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.

Mining the ChEMBL database: an efficient chemoinformatics workflow for assembling an ion channel-focused screening library.

The ChEMBL database was mined to efficiently assemble an ion channel-focused screening library. The compiled library consists of 3241 compounds representing 123 templates across nine ion channel categories. Compounds in the screening library are annotated with their respective ion channel category to facilitate back-tracing of prospective molecular targets from phenotypic screening results. The established workflow is adaptable to the construction of focused screening libraries for other therapeutic target classes with diverse recognition motifs.

C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays.

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 µM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.

Privileged Structures and Polypharmacology within and between Protein Families.

Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries.

On the origins of three-dimensionality in drug-like molecules.

AIM: Many medicinal chemistry-relevant structures and core scaffolds tend toward geometric planarity, which hampers the optimization of physicochemical properties desirable in drug-like molecules. As challenging drug target classes emerge, the exploitation of molecular three-dimensionality in lead optimization is becoming increasingly important. While recent interest has emphasized the importance of enhanced three-dimensionality in molecular fragment designs, the extent to which this is required in core scaffolds remains unclear. MATERIALS & METHODS: Three computational methods, Scaffold Tree deconstruction, Synthetic Disconnection Rules retrosynthetic deconstruction and virtual library enumeration, are applied, together with the descriptors plane of best fit and principal moments of inertia, to investigate the origins of three-dimensionality in drug-like molecules. CONCLUSION: This study informs on the stage at which molecular three-dimensionality should be considered in drug design.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Discovery of biphenylacetamide-derived inhibitors of BACE1 using de novo structure-based molecular design.

ß-Secretase (BACE1), the enzyme responsible for the first and rate-limiting step in the production of amyloid-ß peptides, is an attractive target for the treatment of Alzheimer's disease. In this study, we report the application of the de novo fragment-based molecular design program SPROUT to the discovery of a series of nonpeptide BACE1 inhibitors based upon a biphenylacetamide scaffold. The binding affinity of molecules based upon this designed molecular scaffold was increased from an initial BACE1 IC50 of 323 µM to 27 µM following the synthesis of a library of optimized ligands whose structures were refined using the recently developed SPROUT-HitOpt software. Although a number of inhibitors were found to exhibit cellular toxicity, one compound in the series was found to have useful BACE1 inhibitory activity in a cellular assay with minimal cellular toxicity. This work demonstrates the power of an in silico fragment-based molecular design approach in the discovery of novel BACE1 inhibitors.

Optimisation of the anti-Trypanosoma brucei activity of the opioid agonist U50488.

Screening of the Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC(50) value of 59 nM as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure-activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.