Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report ...the design and characterisation of GSK215, a potent, selective, FAK‐degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS‐4718. X‐ray crystallography revealed the molecular basis of the highly cooperative FAK‐GSK215‐VHL ternary complex, and GSK215 showed differentiated in‐vitro pharmacology compared to VS‐4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long‐lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK‐degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.
A PROTAC with an unusually short linker potently degrades focal adhesion kinase (FAK). SPR and X‐ray crystallography revealed a highly cooperative FAK‐PROTAC‐VCB ternary complex, and FAK degradation showed enhanced effects on 3D cell growth compared to FAK inhibitors. In mice, GSK215 induced rapid and sustained degradation of FAK with a profound PK/PD disconnect.
Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a ...disease‐modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment‐screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment–protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.
PhotoAffinity Bit (PhABit) is a photoreactive fragment‐screening platform to covalently capture fragment–protein interactions. Hits can be profiled and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is widely applicable to novel protein targets, identifying starting points in the development of therapeutics.
There are more H atoms than any other type of atom in an X‐ray crystal structure of a protein–ligand complex, but as H atoms only have one electron they diffract X‐rays weakly and are `hard to see'. ...The positions of many H atoms can be inferred by our chemical knowledge, and such H atoms can be added with confidence in `riding positions'. For some chemical groups, however, there is more ambiguity over the possible hydrogen placements, for example hydroxyls and groups that can exist in multiple protonation states or tautomeric forms. This ambiguity is far from rare, since about 25% of drugs have more than one tautomeric form. This paper focuses on the most common, `prototropic', tautomers, which are isomers that readily interconvert by the exchange of an H atom accompanied by the switch of a single and an adjacent double bond. Hydrogen‐exchange rates and different protonation states of compounds (e.g. buffers) are also briefly discussed. The difference in heavy (non‐H) atom positions between two tautomers can be small, and careful refinement of all possible tautomers may single out the likely bound ligand tautomer. Experimental methods to determine H‐atom positions, such as neutron crystallography, are often technically challenging. Therefore, chemical knowledge and computational approaches are frequently used in conjugation with experimental data to deduce the bound tautomer state. Proton movement is a key feature of many enzymatic reactions, so understanding the orchestration of hydrogen/proton motion is of critical importance to biological chemistry. For example, structural studies have suggested that, just as a chemist may use heat, some enzymes use directional movement to protonate specific O atoms on phosphates to catalyse phosphotransferase reactions. To inhibit `wriggly' enzymes that use movement to effect catalysis, it may be advantageous to have inhibitors that can maintain favourable contacts by adopting different tautomers as the enzyme `wriggles'.
H atoms are `hard to see' in X‐ray crystal structures of protein–ligand complexes. This paper discusses the problem of identifying the correct tautomeric form(s) of protein‐bound ligands.
Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while ...in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry ...have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) ...polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC–MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
Bromodomain-containing proteins are key epigenetic regulators of gene transcription and readers of the histone code. However, the therapeutic benefits of modulating this target class are largely ...unexplored due to the lack of suitable chemical probes. This article describes the generation of lead molecules for the BET bromodomains through screening a fragment set chosen using structural insights and computational approaches. Analysis of 40 BRD2/fragment X-ray complexes highlights both shared and disparate interaction features that may be exploited for affinity and selectivity. Six representative crystal structures are then exemplified in detail. Two of the fragments are completely new bromodomain chemotypes, and three have never before been crystallized in a bromodomain, so our results significantly extend the limited public knowledge-base of crystallographic small molecule/bromodomain interactions. Certain fragments (including paracetamol) bind in a consistent mode to different bromodomains such as CREBBP, suggesting their potential to act as generic bromodomain templates. An important implication is that the bromodomains are not only a phylogenetic family but also a system in which chemical and structural knowledge of one bromodomain gives insights transferrable to others.
Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain “reader” modules. Inhibitors of the ...bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.
The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the ...discovery and structure–activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.
PDF
