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1.  Inhibitors of Tubulin Assembly Identified through Screening a Compound Library 
Chemical biology & drug design  2008;72(6):513-524.
Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 (5), displayed an IC50 of 13 μm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 (8), exhibited good activity against mammalian tubulin (IC50 = 5.0 μm). This compound was also toxic to several cancer cell lines with IC50 values in the region of 1 μM. Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC50 values of 1.1 and 2.8 μM). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC50 values ranging from 0.18 to 0.73 μM.
PMCID: PMC3677961  PMID: 19090918
Tubulin; screen; library; Leishmania
2.  Reversible protein affinity-labelling using bromomaleimide-based reagents† †Electronic supplementary information (ESI) available: 1H and 13C spectra for all new and known compounds, and ES-MS spectra for all reactions with proteins described herein. See DOI: 10.1039/c3ob40239hClick here for additional data file.  
Organic & Biomolecular Chemistry  2013;11(15):2408-2411.
A mild and highly efficient, reversible protein biotinylation method using a hydrolytically stable linker and mild disassembly conditions is described.
Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics.
PMCID: PMC3763775  PMID: 23462873
3.  Evaluating the use of Apo-neocarzinostatin as a cell penetrating protein 
Protein–ligand complex neocarzinostatin (NCS) is a small, thermostable protein-ligand complex that is able to deliver its ligand cargo into live mammalian cells where it induces DNA damage. Apo-NCS is able to functionally display complementarity determining regions loops, and has been hypothesised to act as a cell-penetrating protein, which would make it an ideal scaffold for cell targeting, and subsequent intracellular delivery of small-molecule drugs. In order to evaluate apo-NCS as a cell penetrating protein, we have evaluated the efficiency of its internalisation into live HeLa cells using matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry and fluorescence microscopy. Following incubation of cells with apo-NCS, we observed no evidence of internalisation.
PMCID: PMC3601848  PMID: 23322746
cell internalisation; cell-penetrating protein; drug delivery; Neocarzinostatin; protein–drug conjugate
4.  α-Tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties To Confer Dinitroaniline Resistance ▿ † 
Eukaryotic Cell  2010;9(12):1825-1834.
Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.
PMCID: PMC3008275  PMID: 20870876
5.  Crystal structures of Burkholderia cenocepacia dihydropteroate synthase in the apo-form and complexed with the product 7,8-dihydropteroate 
The enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.
An efficient recombinant protein expression system for DHPS from B. cenocepacia (BcDHPS) was prepared, the dimeric enzyme purified in high yield and crystallized. The structure of the apo-enzyme and the complex with the product 7,8-dihydropteroate have been determined to 2.35 Å and 1.95 Å resolution respectively in distinct orthorhombic crystal forms. The latter represents the first crystal structure of the DHPS-pterin product complex, reveals key interactions involved in ligand binding, and reinforces data generated by other structural studies. Comparisons with orthologues identify plasticity near the substrate-binding pocket and in particular a range of loop conformations that contribute to the architecture of the DHPS active site. These structural data provide a foundation for hit discovery. An intriguing observation, an artifact of the analysis, that of a potential sulfenamide bond within the ligand complex structure is mentioned.
Structural similarities between BcDHPS and orthologues from other Gram-negative species are evident as expected on the basis of a high level of sequence identity. The presence of 7,8-dihydropteroate in the binding site provides details about ligand recognition by the enzyme and the different states of the enzyme allow us to visualize distinct conformational states of loops adjacent to the active site. Improved drugs to combat infections by Burkholderia sp. and related Gram-negative bacteria are sought and our study now provides templates to assist that process and allow us to discuss new ways of inhibiting DHPS.
PMCID: PMC3098144  PMID: 21554707
6.  Synthesis, Biological Evaluation, and Molecular Modeling of 3,5-Substituted-N1-phenyl-N4, N4-di-n-butylsulfanilamides as Antikinetoplastid Antimicrotubule Agents 
Bioorganic & medicinal chemistry  2007;15(18):6071-6079.
Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due the potentially mutagenic nitro groups. Analogues of N1-phenyl-3,5-dinitro-N4,N4-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leishmania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and methyl ketone moieties. Dicyano compound 5 displays IC50 values that are comparable to 2b against purified leishmanial tubulin assembly (6.6 vs. 7.4 μM), Trypanosoma brucei brucei growth in vitro (0.26 vs. 0.18 μM), L. donovani axenic amastigote growth in vitro (4.4 vs. 2.3 μM), and in vitro toxicity against Vero cells (16 vs. 9.7 μM). Computational studies provide a rationale for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions of the sulfanilamide ring.
PMCID: PMC1994923  PMID: 17618122
Leishmania; Trypanosome; Chemotherapy; Tubulin; Dinitroaniline
7.  The architecture of Trypanosoma brucei tubulin-binding cofactor B and implications for function 
The Febs Journal  2013;280(14):3270-3280.
Tubulin-binding cofactor (TBC)-B is implicated in the presentation of α-tubulin ready to polymerize, and at the correct levels to form microtubules. Bioinformatics analyses, including secondary structure prediction, CD, and crystallography, were combined to characterize the molecular architecture of Trypanosoma brucei TBC-B. An efficient recombinant expression system was prepared, material-purified, and characterized by CD. Extensive crystallization screening, allied with the use of limited proteolysis, led to structures of the N-terminal ubiquitin-like and C-terminal cytoskeleton-associated protein with glycine-rich segment domains at 2.35-Å and 1.6-Å resolution, respectively. These are compact globular domains that appear to be linked by a flexible segment. The ubiquitin-like domain contains two lysines that are spatially conserved with residues known to participate in ubiquitinylation, and so may represent a module that, through covalent attachment, regulates the signalling and/or protein degradation associated with the control of microtubule assembly, catastrophe, or function. The TBC-B C-terminal cytoskeleton-associated protein with glycine-rich segment domain, a known tubulin-binding structure, is the only such domain encoded by the T. brucei genome. Interestingly, in the crystal structure, the peptide-binding groove of this domain forms intermolecular contacts with the C-terminus of a symmetry-related molecule, an association that may mimic interactions with the C-terminus of α-tubulin or other physiologically relevant partners. The interaction of TBC-B with the α-tubulin C-terminus may, in particular, protect from post-translational modifications, or simply assist in the shepherding of the protein into polymerization.
PMCID: PMC3806363  PMID: 23627368
CAP-Gly domain; CD; crystallography; tubulin-binding; ubiquitin-like
8.  Cysteine Promoted C-Terminal Hydrazinolysis of Native Peptides and Proteins** 
PMCID: PMC4065347  PMID: 24123371
acyl transfer; erythropoietin; hydrazides; native chemical ligation; peptides

Results 1-8 (8)