Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Paclitaxel and a semi-synthetic analogue docetaxel (Scheme) are two taxane anti-tumor agents that are used against a number of cancers.[1] The taxanes bind to the β-subunit of the dimeric protein α,β-tubulin in microtubules in a 1:1 molar ratio, resulting in a decrease in the dynamic nature of microtubules leading to mitotic arrest and apoptotic cell death.[2] The taxanes also promote the assembly of tubulin into microtubules.[3] Docetaxel is two to three times as effective as paclitaxel in promoting the assembly of mammalian brain tubulin in vitro and has a binding constant that is greater than that of paclitaxel by the same factor.[4] We have been using site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin to examine the taxane binding site in tubulin. Although wild-type S. cerevisiae tubulin does not bind taxanes, we were able to instill taxane binding by making five mutations in β-tubulin.[5] The rationale for choosing the five sites to mutate was based on the electron crystal structure of the mammalian brain tubulin-paclitaxel complex.[6] This structure indicated that the amino acid side chains, K19, V23, D26, H227 and F270, are important in taxane binding. In S. cerevisiae β-tubulin these sites are occupied by different amino acids, A19, T23, G26, N227, and Y270. When we exchanged the five residues for those that occur in brain β-tubulin, yeast tubulin was able to bind paclitaxel.[5] We are currently in the process of determining the relative importance of each residue to taxane binding by systematically reversing our original mutations. As a screen to measure the effects of the changes, we are using a cell-based assay in which we examine effects of the mutations on cell proliferation. To be able to use a cell-based assay we introduced the mutated β-tubulin gene into a yeast strain that has diminished multidrug transport activity[7] to produce a strain that is sensitive to paclitaxel (AD1-8-tax).[8] In the course of these studies we found interesting differences in the sensitivity of the various strains to paclitaxel and docetaxel. Scheme Structures of paclitaxel and docetaxel. The effect of the two taxanes on the growth of the strains is presented in Table 1. The letters B and Y refer to the five residues found in brain and S. cerevisiae tubulin, respectively. The data show that 25 µM of either taxane completely inhibited growth of the strain with brain β-tubulin residues at the five positions (BBBBB), but had no effect on the strain with yeast residues (YYYYY). When positions 23, 26, and 270 were changed back to the residues in yeast β-tubulin, the strains (BYBBB, BBYBB, BBBBY) were insensitive to both taxanes, indicating the importance of these residues to taxane binding. However, substituting a yeast residue at position 19 (YBBBB) or 227 (BBBYB) did not affect the sensitivity to paclitaxel, i.e., the paclitaxel ID50 values were essentially unchanged. On the other hand, the sensitivity of these two strains to docetaxel decreased. The K19A and H227N mutations caused the ID50 for docetaxel to increase approximately 5 to 6-fold. Table 1 Growth inhibition by paclitaxel and docetaxel To further examine the effect of the H227N mutation, tubulin was purified from this strain (BBBYB) as well as from AD1-8-tax (BBBBB) and used in in vitro assembly and binding experiments. Data presented in Fig. 1 demonstrate that assembly was dependent on the taxanes and that, in the case of tubulin from strain BBBBB (Fig. 1A), the EC50 values for paclitaxel and docetaxel were 1.1 µM and 0.36 µM, respectively. However, with tubulin from the H227N (BBBYB) strain, the values were 2.2 µM for paclitaxel and 3.0 µM for docetaxel (Fig. 1B). Thus, the H227N mutation increased the ED50 for docetaxel 8.3-fold but had a much less effect on the ED50 of paclitaxel (2-fold increase). The formation of microtubules in these experiments was verified by electron microscopy (data not shown). The effect of the H227N mutation on the tubulin-docetaxel interaction was also seen in a competition-binding assay. Preformed microtubules were incubated with 5 µM 3H-paclitaxel and a varying concentration of docetaxel. The data in Fig. 2 show that the H227N mutation decreased the interaction of docetaxel with yeast tubulin by a factor of 3 to 4, i.e., a 3 to 4 higher concentration of docetaxel was required to decrease paclitaxel binding by 50%. Figure 1 Effect of the H227N mutation on the assembly of yeast tubulin. The conditions of the experiment are described in the Experimental Section. A, strain BBBBB. B, strain BBBYB. Error bars refer to standard error of the mean. Figure 2 Effect of the H227N mutation on the displacement of 3H-paclitaxel by docetaxel. The conditions of the experiment are described in the Experimental Section. To help explain the consequences of having Asn instead of His at residue 227 on paclitaxel and docetaxel activity, we modeled the T-taxol conformation of bound paclitaxel[6] into the binding sites of the two proteins. The T-taxol conformation appears to be the most widely accepted conformation for tubulin-bound paclitaxel[11–14]. We assumed that the basic conformers for paclitaxel and docetaxel would be largely conserved relative to the T-taxol structure reported in the 1JFF crystal structure for both proteins, and thus constructed preliminary models for each of the four relevant complexes; paclitaxel-BBBBB, docetaxel-BBBBB, paclitaxel-BBBYB, and docetaxel-BBBYB, by simply editing the original crystal structure accordingly and effecting local receptor relaxation in the vicinity of the ligand. From these computations, we derived a purely enthalpic estimate that docetaxel bound more strongly than paclitaxel to the BBBBB tubulin by 0.43 kcal/mol and that paclitaxel bound more strongly than docetaxel to the BBBYB tubulin by 1.94 kcal/mol. The main source of the enthalpy difference in the BBBBB case is van der Waals interactions, which are derived from stronger interactions between the bulkier docetaxel t-butyl group at the 3′-N position with H227 and V23 than are achieved by the longer, thinner phenyl at this position on paclitaxel. In the case of the BBBYB tubulin, our calculations suggest that docetaxel may lose a significant portion of its van der Waals advantage relative to paclitaxel by virtue of the His to Asn mutation (Asn is a poorer lipophile). Paclitaxel, however, might gain a binding advantage to BBBYB tubulin by virtue of stronger H-bonding between its benzamide carbonyl and the side chain N227 NH donor site (2.22 A) than is possible for the carbamate carbonyl of docetaxel (2.48 A) (Fig. 3). The combined effects of the decreased van der Waals interaction of docetaxel and the increased H-bonding of paclitaxel to BBBYB tubulin might thus result in stronger binding by paclitaxel. Figure 3 A molecular model showing the H227 and N227 residues in the tubulin-paclitaxel and tubulin-docetaxel complexes. The position of paclitaxel and residues interacting with it are shown (black) superimposed over docetaxel and residues interacting with it ... In comparing the predicted binding conformers for paclitaxel and docetaxel with BBBBB and YBBBB our calculations suggest that the hydrogen atoms on the Cδ of the K19 side chain approach within 4.0 A of the t-butyl group of docetaxel (a favorable distance from the perspective of van der Waals interactions) but that no K19 atom comes within 6.1 A of any atom on the corresponding paclitaxel benzamide ring. Mutation of the lysine to a smaller alanine might thus remove a favorable lipophilic contact from docetaxel, but suggests very minimal effect on paclitaxel binding. In summary, we have shown that the relative effects of paclitaxel and docetaxel on tubulin can be affected differently by mutations in β-tubulin. The yeast tubulin mutagenesis approach that we have developed is a useful tool in the development of new taxane and other microtubule anti-mitotic agents.