King_information.pdf from Computing exponentially faster: implementing a non-deterministic universal Turing machine using DNA

The theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs). However, no attempt has previously been made to build an NUTM, and their construction has been regarded as impossible. Here, we demonstrate the first physical design of an NUTM. This design is based on Thue string rewriting systems, and thereby avoids the limitations of most previous DNA computing schemes: all the computation is local (simple edits to strings) so there is no need for communication, and there is no need to order operations. The design exploits DNA's ability to replicate to execute an exponential number of computational paths in P time. Each Thue rewriting step is embodied in a DNA edit implemented using a novel combination of polymerase chain reactions and site-directed mutagenesis. We demonstrate that the design works using both computational modelling, and <i>in vitro</i> molecular biology experimentation: the design is thermodynamically favourable, microprogramming can be used to encode arbitrary Thue rules, all classes of Thue rule can be implemented, non-deterministic rule implementation, etc. In an NUTM, the resource limitation is space, which contrasts with classical UTMs and QUTMs where it is time. This fundamental difference enables an NUTM to trade space for time, which is significant for both theoretical computer science and physics. It is also of practical importance, for to quote Richard Feynman ‘there's plenty of room at the bottom’. This means that a desktop DNA NUTM could potentially utilize more processors than all the electronic computers in the world combined, and thereby outperform the World's current fastest supercomputer, while consuming a tiny fraction of its energy

Structure of the BTB Domain of Keap1 and Its Interaction with the Triterpenoid Antagonist CDDO

<div><p>The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.</p></div

Comparison of Keap1 BTB C151W mutant with apo and CDDO-bound structures.

<p>(A) Overlay of Keap1 BTB C151 (white carbons) and C151W mutant (red carbons) in Cys 151 region. The final 2mF_o-DF_c electron density (contoured at 1σ) is shown as a green mesh for the Trp 151 side-chain. (B) Overlay of the C151W BTB mutant (red carbons) and BTB-CDDO (green carbon) showing overlap between the antagonist and indole ring system of tryptophan. The volume occupied by the side-chain of Trp 151 has been highlighted as a white surface.</p

The structure of Keap1 BTB.

<p>(A) Overall fold of the Keap1 BTB crystallographic dimer as a cartoon representation. The N and C-termini, and key alpha-helical secondary structural elements are labelled for one BTB monomer. The approximate position of Cys 151 is marked with an asterisk. (B) Surface around Cys 151 coloured according to electrostatic potential. Blue regions indicate areas of positive potential and red regions areas of negative potential as calculated by AstexViewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098896#pone.0098896-Hartshorn1" target="_blank">[70]</a>. (C) Details of Cys 151 environment showing close contact with Arg 135. Note that the side-chains of Lys 131 and and Lys 150 are highly flexible and exhibit very weak electron density. Some disorder is also evident for Arg 135.</p

Position of Keap1-bound CDDO in the context of possible Cul3 binding surfaces.

<p>(A) Superposition of KLHL11/Cul3 (PDB code 4ap2) and BTB-CDDO showing proximity to Cul3 N-terminal tail (marked with arrow). KLHL11 is shown in a yellow cartoon representation, Cul3 in blue and the Keap1 BTB dimer in red and green. The CDDO binding site is highlighted as a grey surface, with the surface of CDDO shown in green. For clarity, bound CDDO is only shown for one BTB monomer, and a single copy of KLHL11/Cul3 is shown, although the KLHL11/Cul3 complex dimerizes through the KLHL11 BTB domain in the crystal lattice. (B) Surface representation of the KLHL11 Cul3 binding groove coloured by amino acid sequence identity with Keap1 (blue  =  identical, red  =  non-identical). Keap1 and KLHL11 sequences were aligned using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098896#pone.0098896-Larkin1" target="_blank">[71]</a> to determine regions of identity. The Cul3 N-terminal strand is depicted with blue carbons, and CDDO is shown as a surface representation in green. The position of CDDO was taken from that of its complex with BTB when overlaid with the KLHL11/Cul3 structure. The side-chain of Arg 19 is shown truncated to Cβ as deposited with the PDB. (C) Surface representation of BTB in the region of the CDDO binding groove, showing potential alternative path for Cul3 N-terminal tail upon binding to Keap1 (dashed arrow). The Cul3 and the IVR/BACK regions are taken from the superposition of the KLHL11/Cul3 structure with Keap1 BTB.</p

Inhibitory effects of CDDO on Cul3 binding to wild-type C151 and C151S BTB-BACK as measured by AlphaScreen proximity assay.

<p>The change in signal as a function of CDDO concentration is reported relative to a DMSO control, and is the mean of three measurements. Error bars represent the standard deviation.</p

X-ray data collection and refinement statistics.

a<p>R_merge = Σ<i>_h</i>Σ<i>_j</i> |I<i>_h,j</i>−<i>_h</i>|/Σ<i>_h</i>Σ<i>_j</i>|I<i>_h,j</i>|, where I<i>_h,j</i> is the <i>j</i>th observation of reflection <i>h</i>.</p>b<p>R_work = Σ<i>_h||</i>F<i>_oh</i>|−|F<i>_ch</i>||/Σ<i>_h|</i>F<i>_oh</i>|, where F<i>_oh</i> and F<i>_ch</i> are the observed and calculated structure factor amplitudes respectively for the reflection <i>h</i>.</p>c<p>R_free is equivalent to R_work for a 5% subset of reflections not used in the refinement.</p><p>Numbers in parentheses refer to the outer resolution shell.</p

CDDO and its interaction with the Keap1 domain of BTB.

<p>(A) Chemical structure of CDDO/bardoxolone. (B) Surface representation of Keap1 BTB with CDDO bound in region of Cys 151. The final 2mF_o-DF_c electron density (contoured at 1σ) is shown as a green mesh. (C) Overview of CDDO binding in context of the BTB crystallographic dimer (only one binding site is shown for clarity). The surface of the CDDO binding site is shown in grey. (D) Details of covalent and non-covalent interactions between CDDO and BTB. Hydrogen bonds are denoted by dashed lines. The side-chain of Lys 131 is not visible in the electron density and has been truncated to the Cβ atom. (E) Schematic diagram of interactions between CDDO and BTB. His 129 is shown in the protonated state to emphasize its potential to form an electrostatic interaction with the carboxylate of CDDO. (F) Overlay of apo (white carbons) and CDDO-bound BTB (green carbons) in region of Cys 151.</p

Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA₂)

Lp-PLA₂ has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA₂. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC₅₀ > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA₂ inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile

Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA₂) Discovered through X‑ray Fragment Screening

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA₂) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA₂ in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues