Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain: Effects on Domain Dynamics and Internal Flexibility.

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

Myc phosphorylation in its basic helix-loop-helix region destabilizes transient α-helical structures, disrupting Max and DNA binding.

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Mapping Hidden Residual Structure within the Myc bHLH-LZ Domain Using Chemical Denaturant Titration.

Intrinsically disordered proteins (IDPs) underpin biological regulation and hence are highly desirable drug-development targets. NMR is normally the tool of choice for studying the conformational preferences of IDPs, but the association of regions with residual structure into partially collapsed states can lead to poor spectral quality. The bHLH-LZ domain of the oncoprotein Myc is an archetypal example of such behavior. To circumvent spectral limitations, we apply chemical denaturant titration (CDT)-NMR, which exploits the predictable manner in which chemical denaturants disrupt residual structure and the rapid exchange between conformers in IDP ensembles. The secondary structure propensities and tertiary interactions of Myc are determined for all bHLH-LZ residues, including those with poor NMR properties under native conditions. This reveals conformations that are not predictable using existing crystal structures. The CDT-NMR method also maps sites perturbed by the prototype Myc inhibitor, 10058-F4, to areas of residual structure.

Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia.

Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).

Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution.

The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos.

Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A.

Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[2H,15N]-isotope labelling on the C-terminal bromodomain with selective introduction of 13CH3 methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.

Structure guided lead generation for M. tuberculosis thymidylate kinase (Mtb TMK): discovery of 3-cyanopyridone and 1,6-naphthyridin-2-one as potent inhibitors.

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 µM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.

Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation.

In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate-severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a 'molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.

¹H, ¹³C and ¹5N resonance assignment for the human K-Ras at physiological pH.

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(ß), as well as partial H(α), H(ß) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

Prevention of MKK6-dependent activation by binding to p38alpha MAP kinase.

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.