Versatile Lipases from the Candida rugosa-like Family: A Mechanistic Insight Using Computational Approaches.

Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability on sterol esters. Structurally, both kinds of enzymes display an α/ß-hydrolase fold, with a substrate-binding pocket formed by a hydrophobic cavity covered by a mobile lid. However, it has been reported that some lipases from the Candida rugosa-like family display wide substrate specificity on both triglycerides and sterol esters. Among them, enzymes with different biotechnological applications, such as the lipase isoenzymes produced by C. rugosa and the sterol esterase from Ophiostoma piceae, have been exhaustively characterized and their crystal structures are available. Differences in substrate affinity among these proteins have been attributed to changes in their hydrophobicity. In this work, we analyzed the full catalytic mechanisms of these proteins using molecular dynamics tools, gaining insight into their mechanistic properties. In addition, we developed an in silico protocol to predict the substrate specificity using C. rugosa and O. piceae lipases as model enzymes and triglycerides and cholesterol esters with different fatty acid chain lengths as model substrates. The protocol was validated by comparing the in silico results with those described in the literature. These results would be useful to perform virtual screening of substrates for enzymes of the C. rugosa-like family with unknown catalytic properties.

Structural Basis of Noscapine Activation for Tubulin Binding.

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain in vivo models through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A-O-demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism.

Improvement of the Activity of a Fungal Versatile-Lipase Toward Triglycerides: An in silico Mechanistic Description.

Some enzymes that belong to the Candida rugosa-like lipase family (abH03. 01) combine the activities of lipases and sterol esterases. Thus, they can act on water-insoluble carboxylic esters releasing long-chain fatty acids but also on sterol esters, although with different activity and affinity. The differences in the catalytic properties among the proteins of this family are explained by small changes in the hydrophobicity of some regions. One of such versatile enzymes is the sterol esterase/lipase from Ophiostoma piceae (OPE) that acts very efficiently on the two types of substrates. Structurally, OPE is characterized by the presence of a lid formed by a α-helix and two 310-helices rich in hydrophobic amino acids. In this study, the ope gene was modified by directed mutagenesis in order to change specific amino acids in the lid region to modify its structure with the aim of increasing its hydrophobicity. Several recombinant forms of OPE were heterologously produced in Pichia pastoris. In silico molecular dynamics simulations have been used to decipher the mechanistic principles behind the improvements in substrate catalysis. The analyses suggested that the enhanced activity toward hydrophobic substrates such as triglycerides could be due to a better stabilization of the substrate in the lid region as a result of an increased hydrophobicity and an improved topology. These results indicate that in silico simulations can be useful for the optimization of the activity of lipases from the C. rugose-like family for different biotechnological applications.

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites.

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to ß-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of ß-tubulin.

Triazolopyrimidines Are Microtubule-Stabilizing Agents that Bind the Vinca Inhibitor Site of Tubulin.

Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.

Modification of C-seco taxoids through ring tethering and substituent replacement leading to effective agents against tumor drug resistance mediated by ßIII-Tubulin and P-glycoprotein (P-gp) overexpressions.

In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and ßIII-tubulin overexpression. Based on the structure of C-seco taxoid 1 m (IDN5390) which was believed to more selectively interact with ßIII-tubulin than paclitaxel, we prepared a series of C-seco taxoids bearing various 7,9-O-linkages and/or different substituents at C2 and C3' positions. Some of them exhibited much more potent binding affinity to MTs and cytotoxicity than their C-seco parent compounds in drug resistant cells with both mechanisms. SAR analysis indicated that C2 modifications significantly enhanced MT binding but brought ambiguous influence to cytotoxicity whereas 7,9-linkage and C3' modifications enhance cytotoxicity more efficiently than improve MT binding. These observations illustrate a better translation of molecular binding effect to cellular activity by C ring closure and C3' modification than C2 modification in C-seco taxoids.

Synthesis, Biological Profiling and Determination of the Tubulin-Bound Conformation of 12-Aza-Epothilones (Azathilones).

12-Aza-epothilones (azathilones) incorporating quinoline side chains and bearing different N12-substituents have been synthesized via highly efficient RCM-based macrocyclizations. Quinoline-based azathilones with the side chain N-atom in the meta-position to the C15 atom in the macrocycle are highly potent inhibitors of cancer cell growth in vitro. In contrast, shifting the quinoline nitrogen to the position para to C15 leads to a ca. 1000-fold loss in potency. Likewise, the desaturation of the C9-C10 bond in the macrocycle to an E double bond produces a substantial reduction in antiproliferative activity. This is in stark contrast to the effect exerted by the same modification in the natural epothilone macrocycle. The conformation of a representative azathilone bound to α/ß-tubulin heterodimers was determined based on TR-NOE measurements and a model for the posture of the compound in its binding site on ß-tubulin was deduced through a combination of STD measurements and CORCEMA-ST calculations. The tubulin-bound, bioactive conformation of azathilones was found to be overall similar to that of epothilones A and B.

Structural and Biochemical Characterization of the Interaction of Tubulin with Potent Natural Analogues of Podophyllotoxin.

Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5'-desmethoxy-ß-peltatin A methyl ether (3), 7',8'-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1-4 were potent assembly inhibitors, displaying binding to the colchicine site with Kb values ranging from 11.75 to 185.0 × 10(5) M(-1). In contrast, burseranin (5) was not able to inhibit tubulin assembly. From the structural perspective, the ligand-binding epitopes of compounds 1-3 have been mapped using STD-NMR, showing that B and E rings are the major points for interaction with the protein. The obtained results indicate that the inhibition of tubulin assembly of this family of compounds is more effective when there are at least two methoxyl groups at the E ring, along with a trans configuration of the lactone ring in the aryltetralin lignan core.

TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11.

The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabO(RAB1) resides in the Golgi, RabE(RAB11) localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabE(RAB11), but not RabO(RAB1), immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabE(RAB11) in vivo. hypA1(ts) rapidly shifts RabE(RAB11), but not RabO(RAB1), to the cytosol, consistent with HypA(Trs120) being specifically required for RabE(RAB11) activation. Missense mutations rescuing hypA1(ts) at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabO(RAB1) did not, establishing that the essential role of TRAPPII is facilitating RabE(RAB11) nucleotide exchange. In vitro, TRAPPII purified with HypA(Trs120)-S-tag accelerates nucleotide exchange on RabE(RAB11) and, paradoxically, to a lesser yet substantial extent, on RabO(RAB1). Evidence obtained by exploiting hypA1-mediated destabilization of HypA(Trs120)/HypC(Trs130)/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabO(RAB1), which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypA(Trs120) colocalizes with RabE(RAB11), arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi-to-post-Golgi transition.

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances.

We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by P-gp overexpression, by mutations in the ß-tubulin binding site or by overexpression of the highly dynamic ßIII-tubulin isotype. We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on P-gp and ßIII overexpressing cells and on mutant cells. The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power.

Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism.

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αß-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αß-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to ß-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating ß-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.

Free Energy Profile and Kinetics Studies of Paclitaxel Internalization from the Outer to the Inner Wall of Microtubules.

Several pieces of experimental evidence led us to hypothesize that the mechanism of action of paclitaxel (Taxol) could involve a two-steps binding process, with paclitaxel first binding within the outer wall of microtubules and then moving into the inner binding site. In this work, we first used multiply targeted molecular dynamics (MTMD) for steering paclitaxel from the outer toward the inner binding site. This rough trajectory was then submitted to a refinement procedure in the path collective variables space. Paclitaxel binding energy was monitored along the refined pathway, highlighting the relevance of residues belonging to the H6-H7 and the M- loops. Computational results were supported by kinetics studies performed on fluorescent paclitaxel derivatives.

Zampanolide, a potent new microtubule-stabilizing agent, covalently reacts with the taxane luminal site in tubulin α,ß-heterodimers and microtubules.

Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to ß-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,ß-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates ß-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/ß-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/ß-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/ß-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

Modulation of microtubule interprotofilament interactions by modified taxanes.

Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αß-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 ß-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop.