Добірка наукової літератури з теми "Optimisation hit-To-Lead"

Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide–protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson–Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides’ toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein–peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (−9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

A novel flow-based approach for the preparation of benzimidazol-2-one (1) scaffold by the 1,1′-carbonyldiimidazole (CDI)-promoted cyclocarbonylation of o-phenylenediamine (2) is reported. Starting from a preliminary batch screening, the model reaction was successfully translated under flow conditions and optimised by means of design of experiment (DoE). The method allowed the efficient preparation of this privileged scaffold and to set up a general protocol for the multigram-scale preparation in high yield, purity, and productivity, and was successfully applied for the multigram flow synthesis of N-(2-chlorobenzyl)-5-cyano-benzimidazol-2-one, which is a key synthon for hit-to-lead explorations in our anti-inflammatory drug discovery program.

Parasitic worms cause very significant diseases in animals and humans worldwide, and their control is critical to enhance health, well-being and productivity. Due to widespread drug resistance in many parasitic worms of animals globally, there is a major, continuing demand for the discovery and development of anthelmintic drugs for use to control these worms. Here, we established a practical, cost-effective and semi-automated high throughput screening (HTS) assay, which relies on the measurement of motility of larvae of the barber’s pole worm (Haemonchus contortus) using infrared light-interference. Using this assay, we screened 80,500 small molecules and achieved a hit rate of 0.05%. We identified three small molecules that reproducibly inhibited larval motility and/or development (IC50 values of ~4 to 41 µM). Future work will critically assess the potential of selected hits as candidates for subsequent optimisation or repurposing against parasitic nematodes. This HTS assay has a major advantage over most previous assays in that it achieves a ≥ 10-times higher throughput (i.e., 10,000 compounds per week), and is thus suited to the screening of libraries of tens of thousands to hundreds of thousands of compounds for subsequent hit-to-lead optimisation or effective repurposing and development. The current assay should be adaptable to many socioeconomically important parasitic nematodes, including those that cause neglected tropical diseases (NTDs). This aspect is of relevance, given the goals of the World Health Organization (WHO) Roadmap for NTDs 2021–2030, to develop more effective drugs and drug combinations to improve patient outcomes and circumvent the ineffectiveness of some current anthelmintic drugs and possible drug resistance.

Fragment-based drug discovery (FBDD) has become a mainstream technology for the identification of chemical hit matter in drug discovery programs. To date, the food and drug administration has approved four drugs, and over forty compounds are in clinical studies that can trace their origins to a fragment-based screen. The challenges associated with implementing an FBDD approach are many and diverse, ranging from the library design to developing methods for identifying weak affinity compounds. In this article, we give an overview of current progress in fragment library design, fragment to lead optimisation and on the advancement in techniques used for screening. Finally, we will comment on the future opportunities and challenges in this field.

Focal adhesion kinase (FAK) is a tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers. In cancer cells, FAK promotes the progression and metastasis of tumours. In this study, we used structure-based virtual screening to filter a library of more than 210K compounds against the focal adhesion targeting FAK-focal adhesion targeting (FAT) domain to identify 25 virtual hit compounds which were screened in the invasive breast cancer line (MDA-MB-231). Most notably, compound I showed low micromolar antiproliferative activity, as well as antimigratory activity. Moreover, examination in a model of triple negative breast cancer (TNBC), revealed that, despite not effecting FAK phosphorylation, compound I significantly impairs proliferation whilst impairing focal adhesion growth and turnover leading to reduced migration. Further optimisation and synthesis of analogues of the lead compound I using a four-step synthetic procedure was performed, and analogues were assessed for their antiproliferative activity against three breast cancer (MDA-MB-231, T47D, BT474) cell lines and one pancreatic cancer (MIAPaCa2) cell line. Compound 5f was identified as a promising lead compound with IC50 values in the range of 4.59–5.28 μM in MDA-MB-231, T47D, BT474, and MIAPaCa2. Molecular modelling and pharmacokinetic studies provided more insight into the therapeutic features of this new series.

Abstract eIF4E is the rate limiting factor for cap-dependent protein translation, forming an essential part of the translation initiation complex, eIF4F and is a key signalling convergence point for the major oncogenic pathways MAPK and PI3K/AKT/MTOR. eIF4E has been shown to be overexpressed in cancer, which has been associated with poor prognosis and resistance to chemotherapeutics and targeted agents such as mTOR and MAPK inhibitors. eIF4E is therefore a promising target for cancer treatment but has so far been difficult to target with small molecules due to a lack of druggable pockets. Fragment based crystallographic screening is a powerful technique for probing the surface of proteins to identify potentially druggable binding sites. Here, we describe our fragment-based approach and hit optimisation campaign to generate potent compounds binding to eIF4E For the fragment screen, we generated an engineered form of eIF4E, where the N-terminal (residues 1-35) were removed and replaced a short, tethered peptide based on the canonical binding sequence of the eIF4E binding protein 4E-BP1, and screened 1400 fragments using a combination of Xray crystallography and NMR. Several low affinity (mM) fragment hits were identified, binding to both the mRNA cap- binding site and at an additional binding site of unknown functional relevance. Using a combination of biophysical and biochemical assays, subsequent rounds of iterative structure-based drug design of yielded potent lead molecules against both binding sites. For the cap-binding site, lead compounds of low nM potency were generated but due to the polar nature of the binding site, the molecules had very poor permeability properties and did not inhibit cell proliferation. For the non-canonical binding site, lead compounds with potency of 100nM alongside less potent enantiomers were generated which provided useful tools for probing the function of the binding site in vitro. We developed a MSD (Meso-scale discovery) assay to determine that the lead compound and not the inactive enantiomer could disrupt the eIF4E:eIF4G interaction in HeLa cell lysates with an IC50 of 1uM. Using a HeLa extract in vitro translation assay, we could also demonstrate that both the cap site and non-canonical site lead compounds could inhibit cap-dependent but not IRES driven protein translation. This work demonstrates the power of fragment screening to identify novel functional pockets on difficult to drug proteins such as eIF4E. Citation Format: Caroline J Richardson, Mladen Vincovic, Charlotte East, Nicola Wallis, George Ward, Andrew Woodhead. Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A142.

Abstract My presentation will describe our work on the discovery, nonclinical antitumor properties, mechanism of action (MOA), identification of initial target patient population, and early clinical development of NXP-800. We discovered this agent in our academic drug discovery centre at ICR following our initial identification of the ‘bisamide’ hit CCT245232 from a diverse ~200,000-compound library using an innovative, imaging-based (ArrayScan™) phenotypic screen for inhibitors of the HSF1-mediated activation of the heat shock response in U2OS human osteosarcoma cells. Subsequent multiparameter medicinal chemistry optimisation of the bisamide series, containing the N,N′-4-methyl-1,3-phenylenediamide core, focused on establishing the cell-based SAR for both inhibition of cell proliferation and linked HSF-1 mediated HSP72 induction in the sensitive ARID1A-mutant, platinum-resistant SK-OV-3 human ovarian carcinoma cell line. This generated our orally bioavailable, in vivo-active lead compound and chemical tool CCT251236 – exhibiting oral antitumor efficacy in the SK-OV-3 human ovarian cancer xenograft model in nude mice (Cheeseman et al J Med Chem 60 180-201 2017). Final multiparameter optimization yielded the clinical development candidate CCT361814/NXP800 (Pasqua et al J Med Chem 66 5907-36 2023). With the screening hit bisamide compound already active in the single to low double digit nanomolar range, the early focus was on improving kinetic solubility while retaining the desired antiproliferative and linked HSF1 pathway inhibitory activity together with simultaneous optimisation of PK/PD properties to maximize oral antitumour efficacy; finally requiring modification to the fluorobisamide NXP800 to eliminate PGP-mediated efflux and achieve optimal PK/PD, Pharmacologic Audit Trail, antitumor efficacy and tolerability. In view of these properties, its clean safety panel profile, clear therapeutic index and acceptable dose-to-human prediction, NXP800 was progressed into clinical development. Evaluation in a mini-panel of human cancer cell lines and tumor xenografts revealed high sensitivity to NXP800 in ARIDIA-deficient human ovarian cancer models, confirmed in the large Sanger cancer cell line panel and in isogenic pair systems. Activity exceeded that of cisplatin in both platinum-sensitive and platinum-resistant ARID1A-mutant ovarian cancer xenograft models. By RNA-Seq we identified two sets of consistent, major gene expression profile changes in human cancer cell lines exposed to NXP800, namely: 1) the expected changes in HSF1-regulated genes, together with 2) activation of the integrated stress response (ISR), including ATF4-regulated gene expression. This latter did not indicate a global stress response to NXP800 as we saw no activation of the unfolded protein response (UPR). Physiologically, ISR activation is mediated by phosphorylation of EIF2α which is tightly regulated by four stress-controlled kinases, GCN2, HRI, PKR and PERK. Using each of systematic siRNA knockdown or pharmacologic inhibition by two small-molecule tool compounds from different chemotypes, we discovered that GCN2 alone was necessary and sufficient for ISR activation and cell growth inhibition by NXP800 in SK-OV-3 ovarian cancer cells. Also, orthogonal siRNA depletion or pharmacologic inhibition of GCN2 both markedly reduced the antiproliferative activity of NXP800 in these cells. Global phospho-proteome analysis demonstrated defined changes in response to NXP800 which were reversed by co-treatment with a GCN2 inhibitor. Other follow-on studies at the protein level demonstrated that pharmacological concentrations of NXP800 induced GCN2-dependent phosphorylation of EIF2α, inhibition of global cap-dependent protein translation, and selective translation of ATF4 – a transcription factor that activates downstream genes such as CHAC1 and CHOP in the ISR – both in ARID1A-mutant human ovarian cells in vitro and in corresponding tumor xenograft models in vivo. Using siRNA silencing, we showed that prevention of ATF4 induction substantially reduced the antiproliferative response of SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. Controls used in these experiments included a closely matched, inactive regioisomer. Furthermore, we showed that ISR induction by NXP800 inhibited HSF1 activation, in SK-OV-3 cells, confirming the mechanistic link between ISR activation and inhibition of HSF1-mediated transcription. We further showed that GCN2 activation was not due to the canonical mechanism of amino acid starvation. In summary, we used an HSF1 pathway-focused phenotypic screen to discover the mechanistically novel, first-in-class drug NXP800, which acts potently on ARID1A-mutant, human ovarian cancer cells to stimulate GCN2 and thereby activate the ISR pathway, leading to ATF4 induction and inhibition of HSF1-mediated gene transcription in ARID1A-mutant, platinum-resistant human ovarian cancer cells. Through this mechanism, NXP800 shows highly promising activity in models of this cancer type, including potent inhibition of proliferation in vitro and substantial regression of tumor xenografts in vivo. Studies are currently underway to determine precisely how NXP800 stimulates GCN2 activity and the role of ARID1A deficiency in its pharmacological effects. Recent ChIP-Seq studies are informing on the molecular interactions between ARID1A mutation and the expression of ISR/ATF4 and HSF1-regulated genes. With Nuvectis Pharma, the Phase 1a dose escalation study has been completed and a multicentre Phase 1b expansion cohort study in ARID1A-mutated, platinum-resistant ovarian cancer – a high unmet medical need – has been initiated (NCT05226507) in collaboration with the GOG Foundation and the European Network of Gynaecological Oncological Trial Group (ENGOT). FDA has issued a Fast Track designation to NXP800 in this setting. In addition, NXP800 shows therapeutic potential in non-clinical models of endometrial, gastro-intestinal and bile duct cancer, with FDA Orphan Drug status granted in the last of these indications. Citation Format: Paul Workman. From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA014.

Les travaux réalisés durant cette thèse ont été scindés en trois parties distinctes, s'inscrivant toutes dans le cadre du développement de composés à visée thérapeutique. La première partie de ce manuscrit est consacrée à l'identification d'inducteurs de SLPI (Secretory Leucocyte Protease Inhibitor), une protéine principalement produite par les cellules immunitaires et épithéliales, et possédant de nombreuses propriétés biologiques prometteuses. Un criblage phénotypique a donc été mis en œuvre à partir de deux chimiothèques académiques et a mené à l'identification de 5 hits, capables d'induire la production de SLPI par les lymphocytes B. Ces travaux ont donc permis d'ouvrir la voie aux applications thérapeutiques potentielles de ces composés, notamment dans le cadre du développement de traitements antibiotiques ou anti-inflammatoires. La deuxième partie de ce manuscrit est consacrée à l'identification d'un pharmacophore d'inducteurs d'Interleukine-10 (IL-10), une cytokine produite par les cellules immunitaires, et ayant pour rôle principal la régulation de l'immunité. Préliminairement à ces travaux de thèse, un criblage phénotypique a permis d'identifier des inducteurs de la production d'IL-10 par les lymphocytes B à des fins de thérapie cellulaire visant à réduire les symptômes de la sclérose en plaque. Les travaux réalisés dans le cadre de cette thèse ont donc consisté en l'optimisation hit-to-lead du composé le plus prometteur, afin d'identifier un pharmacophore d'inducteurs d'IL-10, via l'établissement de relations structure-activité robustes. Durant ces travaux, 240 analogues ont été synthétisés. Enfin, la troisième partie de ce manuscrit est consacrée au développement d'une méthodologie de synthèse par voie photochimique, pour l'accès à des composés hétérocycliques originaux. Cette méthodologie ne nécessitant ni chauffage, ni catalyseur métallique, s'inscrit donc pleinement dans le développement d'une chimie plus verte
The work carried out during this thesis has been divided into three distincts parts, all relating to the developpement of compounds for therapeutic use. The first part of this manuscript is devoted to the identification of inducers of Secretory Leucocyte Protease Inhibitor (SLPI), a protein mainly produced by immune and epithelial cells, with numerous promising biological properties. A phenotypic screening was conducted using two academic chemical libraries, leading to the identification of 5 hits able of inducing SLPI production by B cells. This work paved the way for potential therapeutic applications of these compounds, particularly in the development of antibiotic or anti-inflammatory treatments. The second part of this manuscript is dedicated to identifying a pharmacophore for inducers of Interleukin-10 (IL-10), a cytokine produced by immune cells, whose main role is the regulation of immunity. Prior to this thesis, a phenotypic screening was conducted to identifiy inducers of IL-10 production by B cells for use in cell therapy to reduce symptoms of multiple sclerosis. This thesis work involved hit-to-lead optimization of the most promising compound to identify a pharmacophore for IL-10 inducers through the establishment of robust structure-activity relationships. During this phase, 240 analogs were synthetized. Finally, the third part of this manuscript focuses on the development of a photochemical synthesis methology for accessing original heterocylic compounds. This methology, requiring neither heating nor metallic catalysts, aligns with the development of greener chemistry

Les protéines BET, modules impliqués dans la régulation épigénétique, jouent un rôle essentiel dans le développement du cancer. Actuellement plusieurs inhibiteurs de protéines BET font l'objet d'essais cliniques pour le traitement de différents types de cancer. Une des limitations au développement clinique est l'impact général de la modulation génique induite par des inhibiteurs de type ‘pan-BET’ non sélectifs. Un ciblage individuel des protéines BET et la discrimination des domaines BD1 et BD2 conduiraient à un effet transcriptionnel plus spécifique limitant les effets secondaires et l’apparition de résistance. Le criblage d’une chimiothèque focalisée sur les interactions protéine-protéine a permis d’identifier deux molécules avec des profils de sélectivité uniques. Une étude de SAR a révélé le fragment minimal nécessaire à l’interaction ligand-protéine. La résolution de la structure cristallographique en complexe avec Brd4(BD1) a permis de valider nos interprétations et de développer des inhibiteurs BET plus puissants et sélectifs. Une stratégie de synthèse originale orientée vers une diversité structurale (DOTS) combinant le criblage virtuel et l’élaboration automatisée de bibliothèques focalisées a été utilisée. Ce travail a découvert un inhibiteur optimisé de BD1 avec une affinité de 100x supérieure à la molécule initiale et un ratio de sélectivité BD1 vs BD2 égale à 300. L’activité cellulaire d’inhibition du pro-oncogène c-Myc, au µM, a permis de valider le composé en tant que sonde moléculaire. Des études in vitro et in vivo permettront d'élucider le rôle biologique individuel de chaque BD et de valider l'intérêt de leurs développement en clinique
BET-proteins, acting as epigenetic readers, play an essential role in cancer development. To date, numerous potent inhibitors disrupting BET functions have been discovered, including several of them that are undergoing clinical trials for the treatment of different types of cancer. The common drawback limiting their use in clinical practice is an inability to distinguish between BET-members that may cause side effects and resistances. The selective targeting of individual BET and the discrimination between BD1 and BD2 present an opportunity to achieve more selective transcriptional effect. A midthroughput screening of previously designed chemical library allowed identification of two molecules with unique profiles of selectivity that have never been observed. An undertaken structure-based program revealed a minimum scaffolds necessary for binding. Taking together with resolved X-Ray structures it allowed the development of more potent and selective BET inhibitors by DOTS (diversity oriented target focused synthesis) strategy, combining virtual screening and diversity oriented library design. This optimization led to a potent inhibitor with up to 100-fold improvement of affinity to the target and up to 300-fold selectivity toward BD1. Dose-response downregulation of c-Myc levels in low micromolar range in cell assays allowed the validation of the identified molecule as a chemical probe. Further comprehensive in vitro and in vivo evaluations of this compound will enable elucidating the biological role of each bromodomain and a validation of the interest toward the development of selective inhibitors in clinic

The lead generation phase is the stage in early drug discovery that refers to the process of identifying hit molecules that interact with the desired target, followed by a limited optimisation of those hits. During this period the project aims to identify high-quality chemical tools suitable for advanced cellular and in vivo studies which will help to strengthen the validation of the target and increase confidence in the proposed therapeutic hypothesis. This chapter gives an overview of the major hit finding approaches together with their strengths and limitations. The process of hit profiling and hit selection, as well as hit expansion, are illustrated, and complications that can be encountered during this initial phase are highlighted. The advantage of using an integrated lead generation strategy is emphasised. A well-defined lead generation strategy, where two or more complementary hit finding approaches are executed in parallel, combined with a screening cascade containing relevant biological assays, will often result in the identification of structurally diverse lead series, leading ultimately to successful clinical candidates.

Lead Optimisation (LO) is a critical part of the drug discovery process as it is the part where all efforts that go into earlier parts of the process (Target Identification, HTS, Hit-to-Lead, Lead Identification) are crystallised into a single compound, the candidate drug. The optimisation phase is like a treasure hunt, and discovery projects can take many paths to discover their treasured molecule. In this chapter we have chosen to focus on the practicalities of how the LO project can be organised, and what different property issues the project may face as it attempts to find an efficient path forward to find a suitable candidate drug for development. This multiparameter optimisation dance can have many issues, often interconnected. We discuss approaches to many of these problems and offer advice on how project members should think about tackling issues in a holistic way. In this way we hope to support projects in understanding the right issues to tackle at the right time. We offer some tips, and examples, of successes, and suggest that identifying the issue to tackle is key to establishing robust hypotheses to test and so drive projects forward. Our goal in this chapter is to aid project thinking about the best way forward.