Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules.

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking.

Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors.

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >10 -translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors.

Fragment-Based Discovery of Non-bisphosphonate Binders of Trypanosoma brucei Farnesyl Pyrophosphate Synthase.

Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their poor pharmacokinetic properties, they are not well suited for antiparasitic therapy.

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding.

A segmented flow platform for on-demand medicinal chemistry and compound synthesis in oscillating droplets.

We report an automated flow chemistry platform that can efficiently perform a wide range of chemistries, including single/multi-phase and single/multi-step, with a reaction volume of just 14 μL. The breadth of compatible chemistries is successfully demonstrated and the desired products are characterized, isolated, and collected online by preparative HPLC/MS/ELSD.

The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML.

Bioorthogonal Probes for the Study of MDM2-p53 Inhibitors in Cells and Development of High-Content Screening Assays for Drug Discovery.

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high-content screening assays. By using automated image analysis with single-cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. Additionally, we developed a high-throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter.

A General Strategy for Targeting Drugs to Bone.

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS).

Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology.

Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor.

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3.

Large scale meta-analysis of fragment-based screening campaigns: privileged fragments and complementary technologies.

A first step in fragment-based drug discovery (FBDD) often entails a fragment-based screen (FBS) to identify fragment "hits." However, the integration of conflicting results from orthogonal screens remains a challenge. Here we present a meta-analysis of 35 fragment-based campaigns at Novartis, which employed a generic 1400-fragment library against diverse target families using various biophysical and biochemical techniques.

A novel class of oral direct renin inhibitors: highly potent 3,5-disubstituted piperidines bearing a tricyclic p3-p1 pharmacophore.

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay.

Towards intracellular delivery of peptides.

In view of our more comprehensive understanding with respect to intracellular pathways and their regulation, a vast number of interesting drug targets appear to be in a space that can be addressed neither by small molecules nor by biologics. Especially, interference with intracellular protein-protein interactions, if successful, seems to offer considerable opportunities to expand the application of peptides as therapeutics, in particular in the field of oncology. This review focuses on requirements for the development of peptide therapeutics aiming at intracellular targets.

The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: a proof-of-concept study.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia.

Substrate profiling of IGF-1R and InsR: identification of a potent pentamer substrate.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time.

Recent trends and observations in the design of high-quality screening collections.

The design of a high-quality screening collection is of utmost importance for the early drug-discovery process and provides, in combination with high-quality assay systems, the foundation of future discoveries. Herein, we review recent trends and observations to successfully expand the access to bioactive chemical space, including the feedback from hit assessment interviews of high-throughput screening campaigns; recent successes with chemogenomics target family approaches, the identification of new relevant target/domain families, diversity-oriented synthesis and new emerging compound classes, and non-classical approaches, such as fragment-based screening and DNA-encoded chemical libraries. The role of in silico library design approaches are emphasized.

Safe and reliable synthesis of diazoketones and quinoxalines in a continuous flow reactor.

A flow method for the synthesis of aliphatic and aromatic diazoketones from acyl chloride precursors has been developed and used to prepare quinoxalines in a multistep sequence without isolation of the potentially explosive diazoketone. The protocol showcases an efficient in-line purification using supported scavengers with time-saving and safety benefits and in particular a reduction in the operator's exposure to carcinogenic phenylenediamines.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors.

Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity.

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors.

An efficient protocol for the solid-phase synthesis of malondiamides.

A novel and straightforward solid-phase synthesis of malondiamides containing a free nitrogen has been developed. These intermediates, which can be directly obtained in good yield and purity, can be further derivatised. This approach can be used for the synthesis of large split-and-mix-libraries.

Library design for fragment based screening.

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.