Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity.

BACKGROUND: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. METHODS: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. RESULTS: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. CONCLUSIONS: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Discovery of an MLLT1/3 YEATS Domain Chemical Probe.

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine-binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small-molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3-histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small-molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first-in-class probe molecule can be used to understand MLLT1/3-associated biology and the therapeutic potential of small-molecule YD inhibitors.

Open and compressed conformations of Francisella tularensis ClpP.

Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder-order transition of a ß-strand α-helix combination. This transition occurs at a subunit-subunit interface. Our study may now underpin future efforts in a structure-based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188-194. © 2016 Wiley Periodicals, Inc.

Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile.

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+-dependent enzymes, there is little scope to increase co-ordination number during catalysis. We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.

Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.

Discovery of novel immunopharmacological ligands targeting the IL-17 inflammatory pathway.

Interleukin 17 (IL-17) is a proinflammatory cytokine that acts as an immune checkpoint for several autoimmune diseases. Therapeutic neutralizing antibodies that target this cytokine have demonstrated clinical efficacy in psoriasis. However, biologics have limitations such as their high cost and their lack of oral bioavailability. Thus, it is necessary to expand the therapeutic options for this IL-17A/IL-17RA pathway, applying novel drug discovery methods to find effective small molecules. In this work, we combined biophysical and cell-based assays with structure-based docking to find novel ligands that target this pathway. First, a virtual screening of our chemical library of 60000 compounds was used to identify 67 potential ligands of IL-17A and IL-17RA. We developed a biophysical label-free binding assay to determine interactions with the extracellular domain of IL-17RA. Two molecules (CBG040591 and CBG060392) with quinazolinone and pyrrolidinedione chemical scaffolds, respectively, were confirmed as ligands of IL-17RA with micromolar affinity. The anti-inflammatory activity of these ligands as cytokine-release inhibitors was evaluated in human keratinocytes. Both ligands inhibited the release of chemokines mediated by IL-17A, with an IC50 of 20.9 ± 12.6 µM and 23.6 ± 11.8 µM for CCL20 and an IC50 of 26.7 ± 13.1 µM and 45.3 ± 13.0 µM for CXCL8. Hence, they blocked IL-17A proinflammatory activity, which is consistent with the inhibition of the signalling of the IL-17A receptor by ligand CBG060392. Therefore, we identified two novel immunopharmacological ligands targeting the IL-17A/IL-17RA pathway with antiinflammatory efficacy that can be promising tools for a drug discovery program for psoriasis.

Re-Evaluating the Mechanism of Action of α,ß-Unsaturated Carbonyl DUB Inhibitors b-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Cross-linking with Michael Acceptor Motif-Containing Drugs.

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.

Demonstration of the utility of DOS-derived fragment libraries for rapid hit derivatisation in a multidirectional fashion.

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10-14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.

Deliberately Losing Control of C-H Activation Processes in the Design of Small-Molecule-Fragment Arrays Targeting Peroxisomal Metabolism.

Combined photochemical arylation, "nuisance effect" (SN Ar) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein-ligand structure determination. Reactions were deliberately allowed to run "out of control" in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with SN Ar processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.

Burkholderia pseudomallei d-alanine-d-alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target.

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug-resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d-alanine-d-alanine ligase (BpDdl), required for the ATP-assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+d-Ala-d-Ala), and comparisons with orthologues enzymes suggest a ligand-induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow-up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches.

Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design.

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains.

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action.

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.