Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy

Pretubulysin : a new option for the treatment of metastatic cancer

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF(Fbw7) E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer

Immunoblot analysis of tubulysin A- or pretubulysin-treated L3.6pl pancreatic cancer cells.

<p>The cells were treated for the indicated time points with 10 nM tubulysin A or 10 nM pretubulysin. Lysates were immunoblotted for <b>A</b>) Bcl-2 and phosphorylated Bcl-2 (pBcl-2) or <b>B</b>) for PARP; β-actin served as a reference for the quantification of the protein levels. Ctrl.: lysates of untreated control cells.</p

Typical cellular events upon pretubulysin treatment.

<p><b>A</b>) Isolated DNA of human promyelocytic HL-60 cells treated with pretubulysin (1: control, 2: 30 nM, 24 h; 3: 75 nM, 24 h; 4: 30 nM, 48 h; 5: 75 nM, 48 h; 6: standard 1000 bp ladder; 7: standard 100 bp ladder). <b>B</b>) Human U-2 OS osteosarcoma cells that were left untreated (upper panel) or were treated for 24 h with pretubulysin (lower panel). Microtubules are visualized by immunofluorescence staining of α-tubulin (pseudocolor green), and nuclei were stained with Hoechst 33342 (pseudocolor blue). The arrows indicate the abnormal accumulation of microtubules in cells after treatment with 25 nM pretubulysin; fragmented nuclei, and completely depolymerized microtubules were observed at a drug concentration of 50 nM.</p

Induction of apoptosis in L3.6pl cells.

<p>The cells were treated for 48 h with <b>A</b>) varying concentrations of tubulysin A or pretubulysin (n = 2) or <b>B</b>) with 10 nM drug in conjunction with 10 µM caspase inhibitor Q-VD-OPh (n = 3). The percentages of apoptotic cells in the whole cell population were determined by PI staining (*p≤0.05 Anova/Dunnett).</p

HC analysis of the standard deviation of tubulin fluorescence.

<p>Representative HCS results of several experiments on tubulin depolymerization. The SD is used as a proxy for the tubulin distribution within the cytoplasmic segment of U-2 OS osteosarcoma cells treated for 24 h with antimitotic drugs at varying concentrations. Arrows indicate peak SD values for depolymerizing agents due to the higher density of microtubules at intermediate drug concentrations. Bars represent the mean ± SEM of all cellular segments within a well.</p

Tubulysin A and pretubulysin inhibit clonogenic survival of L3.6pl cells.

<p>L3.6pl cells were stimulated for 4 h and then seeded at low confluence. After 6 d, the cells were stained with crystal violet, and the absorbance was measured at 550 nm (n = 3; *p≤0.05 Anova/Dunnett).</p

Structural comparison of tubulysin A (R = OH), tubulysin D (R = H), pretubulysin and a synthetic precursor.

<p>The modifications of all synthetic precursors of pretubulysin (I–VIII) are highlighted [I: Mep replaced by <i>N</i>-methylsarcosine; II: desmethyl Tup variant; III: missing propionate of Tup; IV: MepIleTuv; V: MepIleMe'Val' (γ-amino acid); VI: MepIleMeVal; VII: MepIle; VIII: Mep].</p

In vitro inhibition of tubulin polymerization by tubulysin A, pretubulysin and a small precursor of pretubulysin.

<p><b>A</b>) As a reference, 1 µM tubulysin A was added to a preparation of 10 µM tubulin, and the absorbance was measured over time at 350 nm; the maximum value of the untreated control was set to 100%. Electron micrographs of the corresponding samples are shown on the right. <b>B</b>) Pretubulysin was either added pre-assembly (left panel) or post-assembly (right panel; the arrow indicates the time at which the drug was added) to a preparation of 10 µM tubulin, and the absorbance was measured at 350 nm; the maximum value of the untreated control was set to 100%. The electron micrographs in the upper corners display different degrees of disruption for both treatment types caused by pretubulysin. <b>C</b>) Tubulin protein was treated pre-assembly with either pretubulysin or its precursor, and the absorbance at 350 nm was measured and then expressed relative to the absorbance of the tubulin control (100%). Electron micrographs show disrupted microtubules upon treatment with pretubulysin or its precursor V. The scale bar on each micrograph is 500 nm.</p

The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1.

The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination