Antimitotic Peptides Research Articles

Self‐Assembled Antimitotic Peptide Vesicle Designed fromα,β‐Tubulin Heterodimer Interface for Anticancer Drug Delivery

AbstractPeptide based anticancer molecule act simultaneously as potent anticancer therapeutic as well as unique drug delivery vehicle for the targeted delivery of often cytotoxic abysmally bioavailable anticancer drugs to their designated organelle. The atypical self‐assembling propensity of peptides gives rise to distinct nanostructures capable of encapsulating various drug payload. Among three different types of cytoskeletons presents in eukaryotes, microtubule plays a quintessential role during the course of cell cycle. Microtubule‐targeting agents continues to be the most unwavering classes of antineoplastic drugs for the treatment of cancer. Any intervention to the dynamic tubulin assembly‐disassembly process will definitely lead to complete perturbation of total cell division process. Several tubulin targeted antimitotic drugs had been designed as well as discovered to disrupt this dynamic nature of tubulin monomers either by inducing extensive polymerization or depolymerization, thereby facilitating overall cell cycle arrest. However, in cancer cells, aberrant mTOR activity causes growing resistance against numerous tubulin targeted drugs inducing metastasization, and simultaneous invasion to new healthy tissues. In recent years, numerous tubulin targeted drugs have been found to have high activity in a combination with mTOR inhibitors like tacrolimus, everolimus (RAD001) etc. but restrained bioavailability, non‐specificity and quick excretion are the major impediments for the successful implementation. Combining the immense importance of protein‐peptide interaction for the development of future anticancer therapeutics as well as self‐assembling propensity of peptides we have taken a unique approach to craft a microtubule targeting antimitotic peptide designed fromα,β‐tubulin heterodimer interface, which promotes bothin vitroandin vivotubulin depolymerization, exhibiting middling toxicity towards MCF7 cells, causing cell cycle arrest. Further AFM images of this β‐sheet forming peptide reveals that this peptide upon self‐assembly give rise to spectacular vesicle structural characteristics which can be used as a vehicle for a combination delivery of Docetaxel and RAD001 in order to enhance their individual therapeutic potency. Encapsulation of propidium iodide and concomitant release studies suggest that Pep‐4 vesicles could be a potential candidate for tubulin targeted sustained release of therapeutics. Here, our designed peptide vesicles are found to capable for the delivery of tubulin targeting drug Docetaxel along with an well‐known mTOR inhibiting drug RAD001 successfully to breast cancer cell line in order to achieve a robust symbiotic effect.

Interaction of diazonamide A with tubulin

[3H]Diazonamide A ([3H]DZA), prepared from the natural product isolated from Diazona angulata, bound to tubulin in larger aberrant assembly products (>500 kDa by sizing HPLC) but not to the αβ-tubulin heterodimer. The binding reaction was rapid, but stoichiometry was low. Stoichiometry was enhanced up to 8-fold by preincubating the tubulin in the reaction mixture prior to adding the [3H]DZA. Although Mg2+ did not affect binding stoichiometry, the cation markedly increased the number of tubulin rings (diameter about 50 nm) observed by negative stain electron microscopy. Bound [3H]DZA did not dissociate from the tubulin oligomers despite extensive column chromatography but did dissociate in the presence of 8 M urea. With preincubated tubulin, a superstoichiometric amount of [3H]DZA appeared to bind to the tubulin oligomeric structures, consistent with observations that neither nonradiolabeled DZA nor DZA analogues inhibited binding of [3H]DZA to the tubulin oligomers. Only weak inhibition of binding was observed with multiple antimitotic compounds. In particular, no inhibition occurred with vinblastine, and the best inhibitors of those examined were dolastatin 10 and cryptophycin 1. We compared the aberrant assembly reaction induced by DZA to those induced by other antimitotic peptides and depsipeptides, in particular dolastatin 10, cryptophycin 1, and hemiasterlin, but the results obtained varied considerably in terms of requirements for maximal reactions, polymer morphology, and inhibitory effects observed with antimitotic compounds.

Targeted Polypeptide–Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis

AbstractTunable protein assemblies not only hold a dominant position in vital biological events but are also a significant theme in supramolecular chemistry. Herein, we demonstrated that the intertubular aggregation of microtubules (MTs) could be efficiently regulated by a synergistic polypeptide–tubulin interaction and host–guest complexation. The benzylimidazolium‐modified antimitotic peptide (BP) could recognize the MTs and concurrently form stable inclusion complexes with avirulent cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) in different binding stoichiometries. The self‐assembling morphology of MTs was converted from fibrous to nanoparticulate aggregates via extensive BP⊂CB[8] cross‐linkage, leading to significant cell apoptosis and tumor ablation in vivo. The targeted (BP⊂CB[8])@MT ternary assembly provides a facile supramolecular method to enhance the protein–protein interactions, which may be developed as a therapy for degenerative diseases, such as cancer.

Targeted Polypeptide-Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis.

Tunable protein assemblies not only hold a dominant position in vital biological events but are also a significant theme in supramolecular chemistry. Herein, we demonstrated that the intertubular aggregation of microtubules (MTs) could be efficiently regulated by a synergistic polypeptide-tubulin interaction and host-guest complexation. The benzylimidazolium-modified antimitotic peptide (BP) could recognize the MTs and concurrently form stable inclusion complexes with avirulent cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) in different binding stoichiometries. The self-assembling morphology of MTs was converted from fibrous to nanoparticulate aggregates via extensive BP⊂CB[8] cross-linkage, leading to significant cell apoptosis and tumor ablation in vivo. The targeted (BP⊂CB[8])@MT ternary assembly provides a facile supramolecular method to enhance the protein-protein interactions, which may be developed as a therapy for degenerative diseases, such as cancer.

Self-Assembly of Antimitotic Peptide at Membranes: Computational and Experimental Investigation

We report the assembly behavior of an antimitotic cell penetrating peptide (CPP) bound with membrane and in solution using experimental and computational techniques. Our study reveals that the antimitotic peptide spontaneously self-assembles and forms a β-sheet-like structure, which is important for cellular entry. Further, we found that this peptide strongly interacts with both liposome membrane and MCF7 membrane. Interestingly, we observed that, during interaction with both lipid membrane and cell membrane, the peptide fluctuates (oscillates) in a similar pattern. The fluctuations in the fluorescence intensity of fluorescein-labeled peptide in the membrane of liposome and cell are attributed to the fluctuation of fluorescein between non-fluorescent neutral form and highly fluorescent dianion form (i.e., prototropic processes). The rate of fluctuation or oscillation is significantly faster at the cell membrane (0.75 s) than that (1.4 s) at the liposome membrane.

Abstract 3703: Superior efficacy of novel albumin-binding auristatin E-based prodrugs compared to auristatin E in a panel of human xenograft models in mice

Abstract Auristatins are highly cytotoxic antimitotic tubulin-binding peptides. Of this family, only Adcetris®, an antibody drug conjugate (ADC) derived from monomethyl auristatin E (MMAE), is approved and marketed. Other auristatins such as dolastatin 10, dolastatin 15, and auristatin PE reached phase 1 and 2 clinical trials, but due to systemic toxicity and low antitumor activity they were discontinued.1 Because of the high cytotoxicity, it is important to deliver these drugs selectively to the tumor avoiding premature release in the blood circulation. We developed an acid-sensitive drug delivery system that uses circulating endogenous serum albumin as a macromolecular carrier. The drug is rapidly bound covalently to albumin upon i.v. injection and, following accumulation in the tumor due to enhanced uptake and retention mechanisms, the highly cytotoxic agent is released in a pH-dependent manner.2 Herein we present in-vivo data of the considerably improved efficacy of two auristatin E derived prodrugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, compared to the parent compound auristatin E. The acid-sensitive hydrazone prodrugs were prepared from the respective carbonyl-containing auristatin E derivatives and the water-solubilizing maleimide-bearing linker Sulf07. Female NMRI nu/nu mice were inoculated subcutaneously with patient- or cell-derived human tumor xenografts (A375, A2780, RXF631, LXFA737) and randomized (n=7-8 per group) with starting tumor volumes in the range of 140-350 mm3. AE-Keto-Sulf07 showed excellent antitumor response over a wide dose range (3.0-6.5 mg/kg twice per week over 3-4 weeks), with optimal dosage at 4.5 mg/kg twice per week over 4 weeks. AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg dosed twice per week over 3-4 weeks or at 3.8 mg/kg dosed once per week over 4 weeks, but caused cumulative skin lesions due to scratching and biting. In contrast, auristatin E was dosed at 0.3 mg/kg twice per week over 3-4 weeks and was only marginally active. In summary, we have shown for the first time that auristatin E derivatives, namely AE-Keto-Sulf07 and AE-Ester-Sulf07, bound to circulating albumin demonstrate promising antitumor efficacy and induce statistically significant long-term partial or complete remission in a panel of human xenograft models in mice. Citation Format: Stephan D. Koester, Lara Pes, Johannes P. Magnusson, Serghei Chercheja, Federico Medda, Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi-Kristin Walter, Khalid Abu Ajaj, Anna Warnecke, Felix Kratz. Superior efficacy of novel albumin-binding auristatin E-based prodrugs compared to auristatin E in a panel of human xenograft models in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3703.

Single functionalized graphene oxide reconstitutes kinesin mediated intracellular cargo transport and delivers multiple cytoskeleton proteins and therapeutic molecules into the cell.

Here, we report a covalently functionalized graphene oxide with Tris-(nitrilo Tris-acetic acid) (TGO), which can reconstitute kinesin mediated intracellular cargo transport, and deliver multiple proteins and therapeutic antimitotic dodecapeptide peptides into the cancer cell.

Antimitotic Peptides and Depsipeptides

Tubulin is the target for an ever increasing number of unusual peptides and depsipeptides that were originally isolated from a wide variety of organisms. Since tubulin is the major component of cellular microtubules, which maintain cell shape in interphase and form the mitotic spindle, most of these compounds are highly toxic to mammalian cells. These peptides and depsipeptides disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis. At the biochemical level, the compounds all inhibit the assembly of tubulin into polymer and, in the cases where it has been studied, strongly suppress microtubule dynamics at low concentrations. In most cases the peptides and depsipeptides inhibit the binding of vinblastine and vincristine to tubulin in a noncompetitive manner, inhibit tubulin-dependent GTP hydrolysis, and interfere with nucleotide turnover at the exchangeable GTP site on beta-tubulin. Most of the peptides and depsipeptides induce tubulin to form oligomers of aberrant morphology, including tubulin rings that vary in diameter depending on the (depsi) peptide under study. The purpose of this review is to give an overview of the cellular, biochemical, in vivo, and SAR aspects of this group of compounds. We also summarize initial efforts by computer modeling to decipher a pharmacophore among the diverse structures of these peptides and depsipeptides.

In silico design of tubulin-targeted antimitotic peptides

Microtubules are polymeric structures formed by the self-assembly of tubulin dimers. The growth and shrinkage of these dynamic arrays have a key role during the cell-proliferation process. This makes tubulin the molecular target of many anticancer drugs currently in use or under clinical trial. Their impressive success is limited by the onset of resistant tumour cells during the treatment, so new resistance-proof molecules need to be developed. Here we use molecular dynamics and free-energy calculations to study the network of interactions that allow microtubule formation. Modelling the protein-protein interface allows us to identify the amino acids responsible for tubulin-tubulin binding and thus to design peptides, which correspond to tubulin subsequences, that interfere with microtubule formation. We show that the application of molecular modelling techniques leads to the identification of peptides that exhibit antitubulin activity both in vitro and in cultured cells.

Antimitotic peptide characterized from soybean: role in protection from cancer?

Cell proliferation, or mitosis, is believed to play a central role in the multistep process of carcinogenesis. The recent identification, cloning, and characterization of a potent antimitotic peptide from soybeans is, therefore, of interest in view of epidemiologic associations between intake of soy products and reduced cancer risk. Several questions must be resolved, however, before the biochemistry and epidemiology can be linked.

Structural insight into the inhibition of tubulin by vinca domain peptide ligands

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.

Cytotoxic Simplified Tubulysin Analogues

An efficient route for the synthesis of the tubulysin family of antimitotic peptides was developed. Simplified tubulysin analogues were synthesized to define the minimum pharmacophore required for cytotoxicity. Simplified tubulysin analogues retain significant cytotoxicity and reveal important preliminary structure-activity relationships.

A missense mutation in Caenorhabditis elegans prohibitin 2 confers an atypical multidrug resistance.

Hemiasterlin is a potent antimitotic peptide that interferes with microtubule dynamics at picomolar concentrations in cell culture. The molecule largely eludes P glycoprotein-mediated drug efflux, and an analog is currently being evaluated in clinical trials as cancer chemotherapy. From a nonclonal genetic screen in Caenorhabditis elegans we isolated eight independent mutants resistant to a synthetic hemiasterlin analog. In one recessive mutant, phb-2(ad2154), a point mutation in prohibitin 2 (E130K) protects worms from drug-induced injury. Data indicate that direct binding of hemiasterlin to prohibitin 2 is unlikely. In fact, C. elegans phb-2(ad2154) was also found to be resistant to numerous other drugs that bind tubulin and to camptothecin, yet this mutant was sensitive to nocodazole and phalloidin. Thus, prohibitin 2 is implicated in a previously uncharacterized pathway of multidrug resistance.

Comparison of the Activities of the Truncated Halichondrin B Analog NSC 707389 (E7389) with Those of the Parent Compound and a Proposed Binding Site on Tubulin

The complex marine natural product halichondrin B was compared with NSC 707389 (E7389), a structurally simplified, synthetic macrocyclic ketone analog, which has been selected for clinical trials in human patients. NSC 707389 was invariably more potent than halichondrin B in its interactions with tubulin. Both compounds inhibited tubulin assembly, inhibited nucleotide exchange on beta-tubulin, and were noncompetitive inhibitors of the binding of radiolabeled vinblastine and dolastatin 10 to tubulin. Neither compound seemed to induce an aberrant tubulin assembly reaction, as occurs with vinblastine (tight spirals) or dolastatin 10 (aggregated rings and spirals). We modeled the two compounds into a shared binding site on tubulin consistent with their biochemical properties. Of the two tubulin structures available, we selected for modeling the complex of a stathmin fragment with two tubulin heterodimers with two bound colchicinoid molecules and a single bound vinblastine between the two heterodimers (Nature (Lond) 435:519-522, 2005). Halichondrin B and NSC 707389 fit snugly between the two heterodimers adjacent to the exchangeable site nucleotide. Fitting the compounds into this site, which was also close to the vinblastine site, resulted in enough movement of amino acid residues at the vinblastine site to cause the latter compound to bind less well to tubulin. The model suggests that halichondrin B and NSC 707389 most likely form highly unstable, small aberrant tubulin polymers rather than the massive stable structures observed with vinca alkaloids and antimitotic peptides.

Mechanism of Action of Tubulysin, an Antimitotic Peptide from Myxobacteria

Tubulysin A is a highly cytotoxic peptide with antimitotic activity that induces depletion of cell microtubules and triggers the apoptotic process. Treated cells accumulated in the G2/M phase. Tubulysin A inhibited tubulin polymerization more efficiently than vinblastine and induced depolymerization of isolated microtubule preparations. Microtubule depolymerization could not be prevented by preincubation with epothilone B and paclitaxel, neither in cell-free systems nor in cell lines. In competition experiments, tubulysin A strongly interfered with the binding of vinblastine to tubulin in a noncompetitive way; the apparent Ki was 3 microM. Electron microscopy investigations showed that tubulysin A induced the formation of rings, double rings, and pinwheel structures. The mode of action of tubulysin A resembled that of peptide antimitotics dolastatin 10, phomopsin A, and hemiasterlin. Efforts are underway to develop this new group of compounds as anticancer drugs.

Two Photoaffinity Analogues of the Tripeptide, Hemiasterlin, Exclusively Label α-Tubulin

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.

Ring Polymers of Tubulin Induced by Binding of Natural Antimitotic Peptides

AbstractA number of natural products with potent antimitotic activity are peptides and depsipeptides that bind to tubulin, provoke depolymerization of microtubules, and induce formation of single layer rings of tubulin dimers. These peptides are all hydrophobic and small relative to tubulin (3‐5 amino acid residues), yet induce rings polymers with properties that can differ significantly in size and self‐association. In addition, these compounds exhibit potent cytotoxicity that varies by several hundred fold from one compound to another. Cryptophycin induces unusually homogeneous rings, composed of eight tubulin dimers, that are stable to dilution at least to nanomolar tubulin concentrations.

Localization of the Antimitotic Peptide and Depsipeptide Binding Site on β-Tubulin

Several naturally occurring peptides and depsipeptides which include the cryptophycins, dolastatin 10, hemiasterlin, and phomopsin A have been found to be potent antimitotic agents, causing cell death at picomolar or low nanomolar concentrations. These compounds inhibit microtubule growth, modulate the dynamics of microtubules, and induce the self-association of tubulin dimers into single-walled rings and spirals. These peptides exhibit mutual competitive inhibition in binding to beta-tubulin, while noncompetitively inhibiting the binding of vinblastine and vincristine to beta-tubulin. Despite the abundance of biochemical information, the details of their molecular interactions with tubulin are not known. In this study, using a combination of molecular dynamics simulations and molecular docking studies, a common binding site for cryptophycin 1, cryptophycin 52, dolastatin 10, hemiasterlin, and phomopsin A on beta-tubulin has been identified. Application of these same methods to alpha-tubulin indicated no interaction between alpha-tubulin and any of the peptides. On the basis of the docking results, a model for the mechanism of microtubule disruption and formation of aberrant nonmicrotubule structures is proposed. Both the active site and mechanism of microtubule depolymerization predictions are in good agreement with experimental findings.

Editorial: Peptides as cancer therapeutics

Of the over one hundred anticancer drugs approved for patient use in the United States, the majority are small molecule cytotoxic drugs. Some are proteins such as interferon, interleukin-2, and monoclonal antibodies; only a handful are peptides. Gonadotropin releasing hormone analogue is an example of an anti-cancer peptide that has been used successfully as first line therapy for metastatic hormonal responsive prostate cancer, and it causes few side effects. It works by suppressing testosterone production which in turn inhibits prostate cancer growth. However, with few exceptions, most of the anticancer agents in use nowadays are rather non-specific and cause significant undesirable side effects on patients. There is accordingly a need for the development of new and effective cancer therapeutics. With the major advances in our basic understanding of oncogenesis and the amazing technologic development in recent years in genomics, proteomics, and drug discovery tools such as combinatorial chemistry, computational chemistry, and high throughput assays, there is genuine hope that more effective and less toxic anti-cancer drugs will be developed in the next decade or two. These drugs are likely to be target specific and less toxic. Although many of these drugs being developed are small molecules or proteins, several are peptides. This issue of Peptide Science highlights some of the recent developments in using peptides as cancer therapeutics. In the first article, Hamel describes the biochemical effects of a number of naturally occurring antimitotic peptides that target tubulin. These compounds are derived from plants, marine animals or microorganisms. They include phomopsins, ustiloxins, dolastatin, cemadotin, crytophysin, hemiasterlin, criamide, vitilevuamide, tubulysin, moroidin, and celogentin. Then, Froidevaux and Eberle outline the biochemistry of somatostatin and its five receptor subtypes. They summarize the literature on the use of unlabeled somatostatin analogues in the treatment of a number of different human cancers, and outline the development of radiolabeled somatostatin analogues as cancer targeting agents. This latter approach leads naturally into the subsequent paper by Lam and colleagues, where the rationale for the development of peptides-rather than monoclonal antibodies-as targeting agents is presented. The literature on using phage display or one-bead one-compound combinatorial peptide libraries to identify cell-surface targeting peptides is reviewed, and applications of these peptides to cancer targeting and cancer diagnostics are detailed. The final paper by Kaumaya and his colleagues surveys the literature on the use of peptides as cancer vaccines in patients. It also describes the ongoing research in the authors' laboratory relating to the use of co-stimulatory molecules and cytokines to augment immune responses. Peptides are likely to play a very important role in the treatment of cancer in the future, particularly in the areas of cell surface targeting and tumor peptide vaccines. As such, we hope readers will find this issue of Peptide Science both topical and stimulating.

Interactions of antimitotic peptides and depsipeptides with tubulin.

Tubulin is the target for an ever increasing number of structurally unusual peptides and depsipeptides isolated from a wide range of organisms. Since tubulin is the subunit protein of microtubules, the compounds are usually potently toxic to mammalian cells. Without exception, these (depsi)peptides disrupt cellular microtubules and prevent spindle formation. This causes cells to accumulate at the G2/M phase of the cell cycle through inhibition of mitosis. In biochemical assays, the compounds inhibit microtubule assembly from tubulin and suppress microtubule dynamics at low concentrations. Most of the (depsi)peptides inhibit the binding of Catharanthus alkaloids to tubulin in a noncompetitive manner, GTP hydrolysis by tubulin, and nucleotide turnover at the exchangeable GTP site on beta-tubulin. In general, the (depsi)peptides induce the formation of tubulin oligomers of aberrant morphology. In all cases tubulin rings appear to be formed, but these rings differ in diameter, depending on the (depsi)peptide present during their formation.