Hybrids of the hemiasterlin analogue taltobulin and the dolastatins are potent antimicrotubule agents

The targeting of microtubules is an important mechanism for cancer chemotherapy. However, there is still a need for improved antimicrotubule agents. A number of seemingly structurally disparate peptidic natural products inhibit tubulin polymerization by binding to a region of the tubulin heterodimer close to the vinca binding site. An analogue of the naturally occurring tripeptide hemiasterlin, taltobulin (HTI-286, 3), has advanced to clinical trials. Structure-activity relationship studies of 3 have revealed critical structural elements necessary for antimicrotubule activity that correspond to comparable groups in the amino terminus tripeptide region of the dolastatins. To investigate the structural relationship between the hemiasterlins and the more complex dolastatins, hybrid compounds composed of 3 and the carboxy terminus dipeptides of dolastatin 10, or the dolastatin 15 analogue cemadotin, were synthesized. The resulting hybrid compounds were potent antimicrotubule agents, thus establishing a structural relationship between the hemiasterlins and the dolastatins. This relationship may be useful in the design of analogues having improved activity in resistant cell lines expressing the P-glycoprotein transporter, for establishing structural relationships with other classes of peptidic antimicrotubule agents, or for modeling studies of the tubulin binding site of these agents.     

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non‐radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine‐, vinblastine‐ and paclitaxel‐binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin‐binding site of 4‐substituted methoxylbenzoyl‐aryl‐thiazoles (SMART‐H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART‐H specifically and reversibly bound only to the colchicine‐binding site, but not to vinblastine‐ or paclitaxel sites. This new non‐radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

Interactions of halichondrin B and eribulin with tubulin

Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that [(3)H]-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K(d) of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of [(3)H]HB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K(i), 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K(i)'s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.     

(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers,blocks taxol binding and results in mitotic arrest

Background: The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail.Results: We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolide's cellular target.Condusions: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.     

Expeditious Total Synthesis of Hemiasterlin through a Convergent Multicomponent Strategy and Its Use in Targeted Cancer Therapeutics

Hemiasterlin is an antimitotic marine natural product with reported sub-nanomolar potency against several cancer cell lines. Herein, we describe an expeditious total synthesis of hemiasterlin featuring a four-component Ugi reaction (Ugi-4CR) as the key step. The convergent synthetic strategy enabled rapid access to taltobulin (HTI-286), a similarly potent synthetic analogue. This short synthetic sequence enabled investigation of both hemiasterlin and taltobulin as cytotoxic payloads in antibody–drug conjugates (ADCs). These novel ADCs displayed sub-nanomolar cytotoxicity against HER2-expressing cancer cells, while showing no activity against antigen-negative cells. This study demonstrates an improved synthetic route to a highly valuable natural product, facilitating further investigation of hemiasterlin and its analogues as potential payloads in targeted therapeutics.     

The anticancer natural product pironetin selectively targets Lys352 of alpha-tubulin

Pironetin is a potent inhibitor of tubulin assembly and arrests cell cycle progression in M phase. Analyses of its structure-activity relationships suggested that pironetin covalently binds tubulin. To determine the binding site of pironetin, we synthesized biotinylated pironetin, which inhibited tubulin assembly both in vitro and in situ. The biotinylated pironetin selectively and covalently bound with tubulin. Partial digestion of biotinylated pironetin-treated tubulin by several proteases revealed that the binding site is the C-terminal portion of alpha-tubulin. By systematic alanine scanning, the pironetin binding site was determined to be Lys352 of alpha-tubulin. Lys352 is located at the entrance of a small pocket of alpha-tubulin, and this pocket faces the beta-tubulin of the next dimer. This is the first compound that covalently binds to the alpha subunit of tubulin and Lys352 of alpha-tubulin and inhibits the interaction of tubulin heterodimers.     

NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.     

Stiff‐Stilbene Photoswitches: From Fundamental Studies to Emergent Applications

Stiff‐stilbene, a sterically restricted fused ring analogue of stilbene, has been regularly used as a model compound in theoretical studies of stilbene photoisomerization. Lately, owing to its excellent photoswitching properties, it is increasingly being applied to reversibly control the properties and function of chemical as well as biological systems. Stiff‐stilbene photoswitches possess a number of advantageous properties including a high quantum yield for photoisomerization and a high thermal stability. Furthermore, they undergo a large geometrical change upon isomerization and their synthesis is straightforward. Herein, we provide an overview of the basic properties of stiff‐stilbene and of recent applications in supramolecular chemistry, catalysis, and biological systems.     

Brazilin Inhibits Prostatic Acidic Phosphatase Fibrillogenesis and Decreases its Cytotoxicity

A 39‐amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP_248–286, is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a “semen‐derived enhancer of virus infection” (SEVI). Therefore, the inhibition of the formation of PAP_248–286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP_248–286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP_248–286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α‐helices and random coils into β‐sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L⁻¹. Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP_248–286 monomer (dissociation constant, 4.03 μmol L⁻¹), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small‐molecule agent against SEVIs.     

Discovery of Novel Diarylamide N-Containing Heterocyclic Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC₅₀ values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.     

Stiff-Stilbene Photoswitches: From Fundamental Studies to Emergent Applications

Stiff-stilbene, a sterically restricted fused ring analogue of stilbene, has been regularly used as a model compound in theoretical studies of stilbene photoisomerization. Lately, owing to its excellent photoswitching properties, it is increasingly being applied to reversibly control the properties and function of chemical as well as biological systems. Stiff-stilbene photoswitches possess a number of advantageous properties including a high quantum yield for photoisomerization and a high thermal stability. Furthermore, they undergo a large geometrical change upon isomerization and their synthesis is straightforward. Herein, we provide an overview of the basic properties of stiff-stilbene and of recent applications in supramolecular chemistry, catalysis, and biological systems.     

The pH-controlled[2]or [3]pseudorotaxanes based on stilbene dye SD⊂CB[7]

We studied characteristics of [2] or [3]pseudorotaxanes based on stilbene dye SD?CB[7] . In neutral or weak alkaline conditions, SD could be encapsulated inside the stilbene site of CB[7] with 1:1 stoichiometry, whereas, with the pH changing to acidity, the hydrogen bonding between the carboxyl group and the carbonyl of CB[7] maked another CB[7] molecule include into hexanoic acid site, these processes were confirmed by fluorescence spectra,UV–Vis spectra and 1H NMR.     

Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors

Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10,000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) A(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate.     

Effect of GTP and GDP nucleotides on vinblastine binding affinity to tubulin: a DFT study

Vinblastine (VLB) is an anticancer agent that inhibits microtubule assembly by binding with tubulin. Density functional theory (DFT) calculations are used to examine low-energy minima of the energy surface of vinblastine-tubulin complex. Thermodynamic data of the binding site of vinblastine to tubulin are extracted with the hybrid DFT (B3LYP (Becke, three-parameter, Lee–Yang–Parr)) method, and then the influence of several solvents, such as water, methanol and ethanol, and different temperatures are discussed on infrared parameters by self-consistent reaction field (SCRF = dipole) method. The effect of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotides on vinblastine binding affinity to tubulin was realised in water solvent by comparing the changes of ?G (Gibbs free energy) of VLB-tubulin and VLB-tubulin bonded to GTP or GDP. The result showed that GDP and GTP increase significantly the binding affinity and the role of GDP is more important than that of GTP.     

The assembly-inducing laulimalide/peloruside a binding site on tubulin: molecular modeling and biochemical studies with [³H]peloruside A

We used synthetic peloruside A for the commercial preparation of [3H]peloruside A. The radiolabeled compound bound to preformed tubulin polymer in amounts stoichiometric with the polymer's tubulin content, with an apparent K(d) value of 0.35 μM. A less active peloruside A analogue, (11-R)-peloruside A and laulimalide acted as competitive inhibitors of the binding of the [3H]peloruside A, with apparent K(i) values of 9.3 and 0.25 μM, respectively. Paclitaxel, epothilone B, and discodermolide had essentially no ability to inhibit [3H]peloruside A binding, confirming that these compounds bind to a different site on tubulin polymer. We modeled both laulimalide and peloruside A into the binding site on β-tubulin that was identified by Huzil et al. (J. Mol. Biol. 2008, 378, 1016-1030), but our model provides a more reasonable structural basis for the protein-ligand interaction. There is a more complete desolvation of the peloruside A ligand and a greater array of favorable hydrophobic and electrostatic interactions exhibited by peloruside A at its β-tubulin binding site. In addition, the protein architecture in our peloruside A binding model was suitable for binding laulimalide. With the generation of both laulimalide and peloruside A binding models, it was possible to delineate the structural basis for the greater activity of laulimalide relative to peloruside A and to rationalize the known structure-activity relationship data for both compounds.     

Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site

The bacterial phosphotriesterase has been utilized as a template for the evolution of improved enzymes for the catalytic decomposition of organophosphate nerve agents. A combinatorial library of active site mutants was constructed by randomizing residues His-254, His-257, and Leu-303. The collection of mutant proteins was screened for the ability to hydrolyze a chromogenic analogue of the most toxic stereoisomer of the chemical warfare agent, soman. The mutant H254G/H257W/L303T catalyzed the hydrolysis of the target substrate nearly 3 orders of magnitude faster than the wild-type enzyme. The X-ray crystal structure was solved in the presence and absence of diisopropyl methyl phosphonate. The mutant enzyme was ligated to an additional divalent cation at the active site that was displaced upon the binding of the substrate analogue inhibitor. These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.     

Interaction between Cytochrome b2 Core and Flavodehydrogenase from the Yeast Hansenula anomala

Abstract— The binding of cytochrome b₂ core (a monomer) to flavodehydrogenase (a tetramer), both purified from Hansenula anomala flavocytochrome b₂, has been studied in the presence of 2- p -toluidinylnaphthalene-6-sulfonate (TNS). The association constant of the TNS-flavodehy-drogenase complex was found to be equal to 0.64 μ M ⁻¹ with a stoichiometry of one TNS per tetramer. Binding of cytochrome b₂ core to flavodehydrogenase was followed by monitoring changes in the TNS fluorescence. Our results indicated that the binding is cooperative, with a stoichiometry of four cytochrome b₂ cores per tetramer of flavodehydrogenase.     

Effect of Temperature on the Cationic Photopolymerization of Epoxides

The effects of temperature on the photoinitiated cationic ring‐opening polymerizations of a number of different epoxide monomers were studied with the aid of a modified optical pyrometer instrument. Depending on the structures of the epoxide monomer, various behaviors were observed. The results were interpreted as due to steric and electronic features inherent in the structures of the monomers that affect the stabilization of the secondary oxonium ions, which are formed as intermediates in these polymerizations. At one extreme, cycloaliphatic epoxides such as cyclohexene oxide give highly reactive oxonium intermediates that exhibit high rates of polymerization even at subambient temperatures. At the other extreme, alkyl glycidyl ethers produce oxonium ion intermediates, which are so stable that they do not spontaneously react to form polymer at room temperature. By manipulation of the structure of the epoxide monomer, novel monomers with tailored reactivities can be prepared.     

Design,synthesis and biological evaluation of isoxazole-naphthalene derivatives as anti-tubulin agents

In this study, a novel series of isoxazole-naphthalene derivatives as tubulin polymerization inhibitors were designed, synthesized and evaluated for their anti-proliferative activities against human breast cancer cell line MCF-7. Most of the synthesized compounds exhibited moderate to potent antiproliferative activity (IC₅₀ < 10.0 μM), as compared to cisplatin (15.24 ± 1.27 μM). Among them, compound 5j containing 4-ethoxy substitution at phenyl ring was found to be the most active compound with IC₅₀ value of 1.23 ± 0.16 μM. Mechanistic studies revealed that compound 5j arrested cell cycle at G2/M phase and induces apoptosis. Furthermore, in vitro tubulin polymerization assay showed that compound 5j displayed better inhibition activity on tubulin polymerization (IC₅₀ = 3.4 μM) than colchicine (IC₅₀ = 7.5 μM). Molecular docking study also revealed that compound 5j binds to the colchicine binding site of tubulin.     

Structural requirements for the interaction of combretastatins with tubulin: how important is the trimethoxy unit?

A series of combretastatins possessing both a trimethoxy unit and other substituents on ring A has been synthesised and tested for cytotoxicity and their ability to interact with the protein tubulin. All previous studies have indicated that the trimethoxy unit is essential for interaction with tubulin. The studies herein show that molecules possessing functionalities other than trimethoxy can also interact with tubulin. Importantly a trimethyl substituted agent 52a has shown reduced cytotoxicity, but increased potency in its ability to inhibit the assembly of tubulin.