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Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.

Mcl-1 is an important cancer target for drug therapy, through which normal apoptosis may be restored by inhibiting its protective function. An scFv and a Fab have been used to generate an apo Mcl-1 crystal system that is amenable to iterative structure-guided drug design., Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

Background and purpose Several anti-angiogenic cancer drugs that inhibit VEGF receptor (VEGFR) signalling for efficacy are associated with a 15-60% incidence of hypertension. Tyrosine kinase inhibitors (TKIs) that have off-target activity at VEGFR-2 may also cause blood pressure elevation as an undesirable side effect. Therefore, the ability to translate VEGFR-2 off-target potency into blood pressure elevation would be useful in development of novel TKIs. Here, we have sought to quantify the relationship between VEGFR-2 inhibition and blood pressure elevation for a range of kinase inhibitors. Experimental approach Porcine aortic endothelial cells overexpressing VEGFR-2 (PAE) were used to determine IC50 for VEGFR-2 phosphorylation. These IC50 values were compared with published reports of exposure attained during clinical use and the corresponding incidence of all-grade hypertension. Unbound average plasma concentration (Cav,u ) was selected to be the most appropriate pharmacokinetic parameter. The pharmacokinetic-pharmacodynamic (PKPD) relationship for blood pressure elevation was investigated for selected kinase inhibitors, using data derived either from clinical papers or from rat telemetry experiments. Key results All-grade hypertension was predominantly observed when the Cav,u was >0.1-fold of the VEGFR-2 (PAE) IC50 . Furthermore, based on the PKPD analysis, an exposure-dependent blood pressure elevation >1 mmHg was observed only when the Cav,u was >0.1-fold of the VEGFR-2 (PAE) IC50 . Conclusions and implications Taken together, these data show that the risk of blood pressure elevation is proportional to the amount of VEGFR-2 inhibition, and a margin of >10-fold between VEGFR-2 IC50 and Cav,u appears to confer a minimal risk of hypertension.

Recent literature has claimed that inhibition of the enzyme MTH1 can eradicate cancer. MTH1 is one of the "housekeeping" enzymes that are responsible for hydrolyzing damaged nucleotides in cells and thus prevent them from being incorporated into DNA. We have developed orthogonal and chemically distinct tool compounds to those published in the literature to allow us to test the hypothesis that inhibition of MTH1 has wide applicability in the treatment of cancer. Here we present the work that led to the discovery of three structurally different series of MTH1 inhibitors with excellent potency, selectivity, and proven target engagement in cells. None of these compounds elicited the reported cellular phenotype, and additional siRNA and CRISPR experiments further support these observations. Critically, the difference between the responses of our highly selective inhibitors and published tool compounds suggests that the effect reported for the latter may be due to off-target cytotoxic effects. As a result, we conclude that the role of MTH1 in carcinogenesis and utility of its inhibition is yet to be established.

MDM2 and MDMX are homologous proteins that bind to p53 and regulate its activity. Both contain three folded domains and ~70% intrinsically disordered regions. Previous detailed structural and biophysical studies have concentrated on the isolated folded domains. The N-terminal domains of both exhibit high affinity for the disordered N-terminal of p53 (p53TAD) and inhibit its transactivation function. Here, we have studied full-length MDMX and found a ~100-fold weaker affinity for p53TAD than does its isolated N-terminal domain. We found from NMR spectroscopy and binding studies that MDMX (but not MDM2) contains a conserved, disordered self-inhibitory element that competes intramolecularly for binding with p53TAD. This motif, which we call the WWW element, is centered around residues Trp200 and Trp201. Deletion or mutation of the element increased binding affinity of MDMX to that of the isolated N-terminal domain level. The self-inhibition of MDMX implies a regulatory, allosteric mechanism of its activity. MDMX rests in a latent state in which its binding activity with p53TAD is masked by autoinhibition. Activation of MDMX would require binding to a regulatory protein. The inhibitory function of the WWW element may explain the oncogenic effects of an alternative splicing variant of MDMX that does not contain the WWW element and is found in some aggressive cancers.

Four poly(ADP-ribose) polymerase (PARP) inhibitors have now presented phase 3 monotherapy data showing compelling benefit of targeting tumours enriched with DNA damage response (DDR) pathway deficiencies, including BRCA gene mutations. Indirect treatment comparisons using the published clinical data from these late stage trials suggest similar levels of monotherapy efficacy are observed in spite of reported differences in PARP trapping potency. However, there is greater diversity in the observed safety profiles. To try and understand these observations, we have carried out a head-to-head comparison of these four PARP inhibitors (olaparib, niraparib, rucaparib and talazoparib) as well as veliparib, which recently reported phase 3 chemotherapy combination data. In our studies, we included an assessment of molecular mechanism of action that included PAR inhibition, PARP trapping and synthetic lethality in isogenic BRCA mutant and wild type models. In addition, an assessment of selectivity in terms of both inhibition of PARP family members using a novel chemoproteomic approach, as well as secondary (off-target) activities was performed. Finally, effects on human haematopoietic stem cell viability and bio-distribution to bone marrow in the rat were tested and compared. A detailed correlation of our datasets with the observed clinical results, including adverse events, suggests these preclinical experiments provide an excellent predictor of clinical response and could be used to assess emerging as well as novel PARP inhibitors. OlaparibVeliparibRucaparibNiraparibTalazoparibCompanyAZAbbVieClovisTesaroPfizerPhaseApprovedIIIApprovedApprovedIIIPARP1 SPR Kd (µM)0.0010.0070.0010.0130.002PARP2 SPR Kd (µM)0.0010.0140.0230.0430.005PARPs with Proteomic Kd Citation Format: Elisabetta Leo, Jeffrey Johannes, Giuditta Illuzzi, Andrew Zhang, Paul Hemsley, Michal J. Bista, Jonathan P. Orme, Verity A. Talbot, Ana J. Narvaez, Elizabeth Underwood, Andrew Pike, Jenni K. Nikkila, Lucy Riches, Sinbad Sweeney, Frida Gustafsson, Anna Cronin, Piero Ricchiuto, Debora A. Roaquin, Fiona Pachl, Eric Miele, Ruth MacDonald, Glen Hawthorne, Andrew N. Mead, Mark J. O'Connor. A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-273.

1,4-Thienodiazepine-2,5-diones have been synthesized via the Ugi-Deprotection-Cyclization (UDC) approach starting from Gewald 2-aminothiophenes in a convergent and versatile manner. The resulting scaffold is unprecedented, cyclic, and peptidomimetic with four points of diversity introduced from readily available starting materials. In addition to eighteen synthesized and characterized compounds, a virtual compound library was generated and evaluated for chemical space distribution and drug-like properties. A small focused compound library of 1,4-thienodiazepine-2,5-diones has been screened for the activity against p53-Mdm2 interaction. Biological evaluations demonstrated that some compounds exhibited promising antagonistic activity.

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

Reactivation of p53 by release of the functional protein from its inhibition by MDM2 provides an efficient, nongenotoxic approach to a wide variety of cancers. We present the cocrystal structures of two complexes of MDM2 with inhibitors based on 6-chloroindole scaffolds. Both molecules bound to a distinct conformational state of MDM2 with nM-μM affinities. In contrast to other structurally characterized antagonists, which mimic three amino acids of p53 (Phe19, Trp23, and Leu26), the compounds induced an additional hydrophobic pocket on the MDM2 surface and unveiled a four-point binding mode. The enlarged interaction interface of the inhibitors resulted in extension of small molecules binding toward the "lid" segment of MDM2 (residues 19-23)-a nascent element that interferes with p53 binding. As supported by protein engineering and molecular dynamics studies, employing these unstable elements of MDM2 provides an efficient and yet unexplored alternative in development of MDM2-p53 association inhibitors.

The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with 13 C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. Unique to NMR is its capability to detect weak microM-mM bindings. NMR assays are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring NMR experiments. We have recently described a time efficient 1D proton NMR assay for studying the effect of antagonists on protein-protein interactions. The method, named AIDA-NMR (for Antagonist Induced Dissociation Assay-NMR), can provide information on whether an antagonist of a protein-protein interaction is strong enough to dissociate the complex and, in addition, whether its interaction is through denaturation, precipitation, or release of a protein in its functional folded state. AIDA requires a large protein fragment (larger than ca. 30 kDa) to bind to a small reporter protein (smaller than ca.12 kDa). Here, we present an extension of this method, named SEI AIDA (SEI, for Selective Excitation-Inversion). The SEI AIDA uses tryptophan-bearing proteins, and by selectively exciting only the proton NMR signals of the (N)H(epsilon) indole side chains of tryptophans, the acquisition time of the AIDA experiment can be reduced by an order of magnitude relative to the corresponding 1D AIDA that uses hard pulses. Thus, at 600 MHz, the (N)H(epsilon) signal of a 35 microM protein complex can be acquired in only 2.5 min, making the SEI AIDA suitable for high-throughput screening pipelines in drug discovery.

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 A. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.

Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.