A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Synthesis and Conformational Analysis of Fluorinated Uridine Analogues Provide Insight into a Neighbouring-Group Participation Mechanism.

Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for 2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the SN1 or SN2 pathway, but instead operates via a neighbouring-group participation mechanism.

Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3'-Deoxy Nucleoside of Pacidamycins.

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

Selectivity of kinase inhibitor fragments.

A kinase-focused screening set of fragments has been assembled and has proved successful for the discovery of ligand-efficient hits against many targets. Here we present some of our general conclusions from this exercise. Notably, we present the first profiling results for literature fragments that have previously been used as starting points for optimization against individual kinases. We consider the importance of screening format and the extent to which selectivity is helpful in selecting fragments for progression. Results are also outlined for fragments targeting the DFG-out conformation and for atypical kinases such as PIM1 and lipid kinases.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amides as potent, selective, inhibitors of JNK2 and JNK3.

The identification and exploration of a novel, potent and selective series of N-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amide inhibitors of JNK2 and JNK3 kinases is described. Compounds 5a and 11a were identified as potent inhibitors of JNK3 (pIC50 6.7 and 6.6, respectively), with essentially equal potency against JNK2 (pIC50 6.5). Selectivity within the mitogen-activated protein kinase (MAPK) family, against JNK1, p38alpha and ERK2, was observed for the series. X-ray crystallography of 5e and 8a in JNK3 revealed a unique binding mode, with the 3-cyano substituent forming an H-bond acceptor interaction with the hinge region of the ATP-binding site.

The identification of potent and selective imidazole-based inhibitors of B-Raf kinase.

A novel triarylimidazole derivative, SB-590885 (33), bearing a 2,3-dihydro-1H-inden-1-one oxime substituent has been identified as a potent and extremely selective inhibitor of B-Raf kinase.

Definition of the heterocyclic pharmacophore of bacterial methionyl tRNA synthetase inhibitors: potent antibacterially active non-quinolone analogues.

Potent inhibitors of bacterial methionyl tRNA synthetase (MRS) have previously been reported. Through SAR of the quinolone moiety, the right hand side pharmacophore for MRS inhibition has now been defined as an NH-C-NH functionality in the context of a bicyclic heteroaromatic system. Potent antibacterial fused-pyrimidone and fused-imidazole analogues have been obtained and enantioselective activity demonstrated. Compound 46 demonstrated very good antibacterial activity against panels of antibiotic-resistant staphylococci and enterococci.

Optimisation of aryl substitution leading to potent methionyl tRNA synthetase inhibitors with excellent gram-positive antibacterial activity.

Optimisation of the left-hand-side aryl moiety of a file compound screening hit against Staphylococcus aureus methionyl tRNA synthetase led to the identification of a series of potent nanomolar inhibitors. The best compounds showed excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics.

The identification of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospholipase A2.

Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency. Cyclopentyl fused derivative 21, SB-480848, showed an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.

The discovery of SB-435495. A potent, orally active inhibitor of lipoprotein-associated phospholipase A(2) for evaluation in man.

The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens.

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.