Thomas P. Garner

Tuneable DNA-based asymmetric catalysis using a G-quadruplex supramolecular assembly

Described is the first modular construction of a G-quadruplex chiral catalyst. The key objective was to use G-quadruplex structures to act as chiral ligands that would allow access to either enantiomer of the product of a Diels-Alder reaction.

Telomestatin: formal total synthesis and cation-mediated interaction of its seco-derivatives with G-quadruplexes.

The structurally unique natural product telomestatin incorporates seven oxazole rings and one sulfur-containing thiazoline in a macrocyclic arrangement. The compound is a potent inhibitor of the enzyme telomerase and therefore provides a structural framework for developing new potential therapeutic agents for cancer. An efficient formal total synthesis of telomestatin is reported in which the key steps are the use of dirhodium(II)-catalyzed reactions of diazocarbonyl compounds to generate six oxazole rings, demonstrating the power of rhodium carbene methodology in organic chemical synthesis. CD spectroscopy establishes that seco-derivatives of telomestatin are potent stabilizers of G-quadruplex structures derived from the human telomeric repeat sequence. Mass spectrometry studies, confirmed by molecular dynamics simulations, provide the first evidence that high affinity binding to terminal G-tetrads in both 1:1 and 2:1 ligand complexes is mediated through the macrocycle coordinating a monovalent cation, with selectivity for the antiparallel structure.

Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappaB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Pagets disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappaB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K(dim) approximately 4-12 microM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappaB luciferase reporter assays, are associated with reduced NF-kappaB activity compared with wt-p62.

Selectivity of small molecule ligands for parallel and anti-parallel DNA G-quadruplex structures

We report CD, ESI-MS and molecular modelling studies of ligand binding interactions with DNA quadruplex structures derived from the human telomeric repeat sequence (h-Tel) and the proto-oncogenic c-kit promoter sequence. These sequences form anti-parallel (both 2 + 2 and 3 + 1) and parallel conformations, respectively, and demonstrate distinctively different degrees of structural plasticity in binding ligands. With h-Tel, we show that an extended heteroaromatic 1,4-triazole (TRZ), designed to exploit pi-stacking interactions and groove-specific contacts, shows some selectivity for parallel folds, however, the polycyclic fluorinated acridinium cation (RHPS4), which is a similarly potent telomerase inhibitor, shows selectivity for anti-parallel conformations implicating favourable interactions with lateral and diagonal loops. In contrast, the unique c-kit parallel-stranded quadruplex shows none of the structural plasticity of h-Tel with either ligand. We show by quantitative ESI-MS analysis that both sequences are able to bind a ligand on either end of the quadruplex. In the case of h-Tel the two sites have similar affinities, however, in the case of the c-kit quadruplex the affinities of the two sites are different and ligand-dependent. We demonstrate that two different small molecule architectures result in significant differences in selectivity for parallel and anti-parallel quadruplex structures that may guide quadruplex targeted drug-design.

Characterization of a non-UBA domain missense mutation of sequestosome 1 (SQSTM1) in Paget's disease of bone.

Mutations affecting the ubiquitin‐associated (UBA) domain of sequestosome 1 (SQSTM1/p62) are commonly found in Pagets disease of bone (PDB) and impair SQSTM1s ability to bind ubiquitin, resulting in dysregulated NF‐κB signaling. In contrast, non‐UBA domain mutations are rarer, and little is known about how they manifest their effects. We present the first characterization at the molecular, cellular, and functional level of a non‐UBA domain missense mutation (A381V) of SQSTM1. Direct sequencing of exon 7 of the SQSTM1 gene in an Italian PDB patient detected a heterozygous C to T transversion at position 1182, resulting in an alanine to valine substitution at codon 381. Pull‐down assays showed the non‐UBA region of SQSTM1 that contains A381 is important in mediating ubiquitin‐binding affinity and that the A381V mutation exerts weak negative effects on ubiquitin binding. Structural and binding analyses of longer UBA constructs containing A381, using NMR spectroscopy and circular dichroism, showed this region of the protein to be largely unstructured and confirmed its contribution to increased ubiquitin‐binding affinity. Co‐transfections of U20S cells showed that the A381V mutant SQSTM1 co‐localized with ubiquitin with a cellular phenotype indistinguishable from wildtype. Finally, effects of the wildtype and mutant SQSTM1 on NF‐κB signaling were assessed in HEK293 cells co‐transfected with an NF‐κB luciferase reporter construct. A381V mutant SQSTM1 produced a level of activation of NF‐κB signaling greater than wildtype and similar to that of UBA domain mutants, indicating that non‐UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF‐κB signaling.

Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.

Independent Interactions of Ubiquitin-Binding Domains in a Ubiquitin-Mediated Ternary Complex

Ubiquitin (Ub) modifications are transduced by receptor proteins that use Ub-binding domains (UBDs) to recognize distinct interaction faces on the Ub surface. We report the nuclear magnetic resonance (NMR) solution structures of the A20-like zinc finger (A20 Znf) UBD of the Ub receptor ZNF216, and its complex with Ub, and show that the binding surface on Ub centered on Asp58 leaves the canonical hydrophobic Ile44 patch free to participate in additional interactions. We have modeled ternary complexes of the different families of UBDs and show that while many are expected to bind competitively to the same Ile44 surface or show steric incompatibility, other combinations (in particular, those involving the A20 Znf domain) are consistent with a single Ub moiety simultaneously participating in multiple interactions with different UBDs. We subsequently demonstrate by NMR that the A20 Znf domain of ZNF216 and the UBA domain of the p62 protein (an Ile44-binding UBD), which function in the same biological pathways, are able to form such a Ub-mediated ternary complex through independent interactions with a single Ub. This work supports an emerging concept of Ub acting as a scaffold to mediate multiprotein complex assembly.

Impact of p62/SQSTM1 UBA Domain Mutations Linked to Paget’s Disease of Bone on Ubiquitin Recognition

The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Pagets disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA-ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.

On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts

Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment.The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant β2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay.Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with β2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities.Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G(3)ACGTAGTG(3))(2) containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G(3)T(4)G(3))(2) dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T(4)-loop sequence. A truncated version of the bimolecular quadruplex d(G(2)ACGTAGTG(2))(2), with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.