Cell free DNA alkylation Assay

The comparably low activity of these analogues in the MTS assay seemed most likely to be the result of poor cell permeability. However, it was also possible that the C-terminal carboxyl group or the amino acid structure itself could have a detrimental impact on DNA alkylation by disrupting minor grove recognition. It was therefore apparent that it would be desirable to ascertain if these compounds could indeed alkylate DNA as expected.

DNA footprinting techniques were deemed most useful to this end, as they would give information not just on the ability of these compounds to alkylate DNA, but also highlight any sequence selectivity they may exhibit.¹⁸⁶ This work was carried out by Prof. Keith R Fox from the University of Southampton, an expert in the sequence specific recognition of DNA by small molecules.

This assay takes advantage of the destabilising effect of DNA alkylation,¹⁸⁷ which results in a strand break upon thermal treatment at the site of covalent attachment of the alkylating agent. The mechanism of strand cleavage is outlined in scheme 3.13. Briefly, alkylation of the nucleobase results in a positively charged adduct. This species destabilises the glycosidic bond between the deoxyribose sugar and the alkylated base.

Thermal treatment accelerates spontaneous cleavage of this bond producing an abasic site in the DNA backbone. The resulting oxocarbenium ion reacts with water to give the cyclic acetal. This species exist in equilibrium with the ring opened aldehyde. The acidic hydrogen vicinal to the carbonyl can be abstracted initiating strand cleavage through the β-elimination of the phosphate group at the 3’ end of the abasic site.

152 In the thermal cleavage assay, the compounds under investigation are incubated with singly end-labelled DNA. In this case the 3’ end is radiolabeled with ³²P. Following incubation the DNA is isolated and thermally treated to induce strand breaks at the site of alkylation. Separation of the DNA fragments based on their relative sized is achieved by denaturing polyacrylamide gel electrophoresis. The gel is then dried and the bands visualised using a phosphorimaging screen, which detects the radioactive signal. As the sequence of DNA is known, the relative position of the bands against a Maxam and Gilbert control ladder, allows identification of the sites of alkylation.

The analogues were tested in the thermal cleavage assay using the MS1 DNA fragment.¹⁸⁸ This is a sequence designed by the Fox group as a general tool for studying the sequence selectivity of minor groove binders, and contains all 136 possible tetranucleotide sequences. The exact sequence of the MS1 fragment can be seen later in figure 3.17.

Figure 3.12 shows the DNA cleavage by the N-acetyl analogues. The compounds were incubated with 1.5 μL (10 nM) of the radiolabelled MS1 fragment at concentrations of 50 μM and 5 µM, overnight at 37 ^OC, in 10 mM Tris-HCl (pH 7.5), containing 10 mM NaCl.

Scheme 3.13 Thermal cleavage at the site of DNA alkylation.

153 Figure 3.12 DNA cleavage by the N-acetyl analogues at incubation concentrations of 50 μM and

5 μM. Lane GA = G+A Maxam and Gilbert ladder. Lane Control = negative control (non-cleaved DNA).

Sample Key:

154 The gel clearly shows that all of the analogues alkylate DNA under these conditions.

There is no apparent difference in sequence selectivity exhibited between the truncated analogues. All appear to alkylate only A as expected, and show a preference only for and thus sterically precludes sufficient minor groove penetration, reducing access to the alkylation site. In addition the narrower minor groove width of AT rich sequences has also been suggested to lead to improved non-covalent binding through stronger van der Waals interactions. Stronger non-covalent binding is advantageous due to the reversible nature of the alkylation event.

As all of the N-acetyl analogues including the ester and acid control compounds, 42 and 43, exhibit essentially identical preference for alkylation sites, it would appear that C-terminal amino acid functionality has no effect on the sequence selectivity of the alkylation event. However it would seem that with the exception of the serine analogue, the C-terminal amino acid group, does appear to have a negative impact on the efficiency of DNA alkylation. This is evident in the differences in the amount of uncleaved DNA remaining at the top of each lane after electrophoresis. It is clear from visual inspection of the gel, that with the exception of the serine analogue, all analogues containing C-terminal amino acid functionality have considerable more uncleaved DNA remaining relative to the ester control 42.

It would be possible to accurately quantify the differences in the remaining uncleaved DNA by measuring the intensity of the radioactive signal. Unfortunately, this was not possible as the observation was made after the results were returned by the Fox group, and this processing could not be performed in house. However, for illustrative purposes, a crude quantitation has been attempted. For this, the image of the gel was expanded, and boxes drawn around the bands of uncleaved DNA remaining in each 50 μM lane. The height of each box was then compared to that of the uncleaved control lane giving an estimation of the percentage of DNA cleaved by each analogue. The results are presented as a bar chart in figure 3.13.

155 The results of this analysis suggest some interesting differences in alkylation efficiency.

However, to confirm these differences as real, it would be necessary to conduct several replicates, and ideally to perform more rigorous kinetic based alkylation assays.

Nevertheless, the observed differences will be discussed.

As the cleavage induced by the carboxylic acid control 43, appears to match that of the ester 42, the detrimental impact of the amino acids is unlikely to be a consequence of the negatively charged carboxylic acid group. Thus the feared potential electrostatic repulsion with the polyanionic backbone of DNA appears not to be the dominant problem.

Therefore, the amino acid structures themselves might be inhibiting the alkylation event.

As discussed, the nature of the side chain may have the potential to mitigate the negative effect of the C-terminal amino acid on DNA alkylation. This is most apparent in the serine analogue 37, which appears to be the only amino acid analogue to have cleaved the same percentage of DNA as the ester control 42_. The serine analogue was also one of the best performing of the amino acid analogues in the MTS assay, breaking the observed trend between hydrophobicity and cellular activity, returning a lower IC50 than the alanine

Figure 3.13 Bar chart of the percentage of DNA cleaved relative to the control lane at 50 μM compound incubation concentration. Estimation of uncleaved DNA band height achieved using the

box method (see below image). Boxes were drawn using Microsoft PowerPoint, and heights measured using the ‘autoshape’ size function.

156 analogue. It is possible that the hydroxyl group is offering an advantage towards DNA alkylation. This will be discussed in more detail later.

The results indicate that the remaining N-acetyl amino acid-duocarmycin conjugates all alkylate DNA with reduced efficiency compared to the ester and acid controls, 42, and 43.

This might be the result of effects on non-covalent minor groove binding. The detrimental impact could result from a number of potential effects, and would likely be the result of a combination of contributing factors.

The most simple explanation may be that the amino acid structure disrupts the planar nature of the compounds and this reduces minor groove binding affinity. In such cases the potential reversal of the alkylation event may be more noticeable, thus reducing consistent with a recent report by Boger et al. that describes a series of increasingly water soluble duocarmycin SA derivatives. These analogues contained varying degrees of PEG substitution at the methoxy groups of the trimethoxyindole subunit. A convincing linear relationship between hydrophilicity and reduced DNA alkylation efficiency was demonstrated.¹⁹⁰

The C-terminal amino acid structures may also impose steric demands which might decrease alkylation efficiency. It has been reported that a simple truncated Boc derivative of the alkylation subunit of CC-1065 (N-Boc-CPI) shows reduced DNA alkylation efficiency when compared to that of duocarmycin (N-Boc-DSA). This has been attributed to a steric effect of the extra indole methyl group of the CPI subunit (see figure 3.14).¹⁸⁹ It is possible that the amino acid structures sterically inhibit the depth of minor grove penetration, or perhaps in some cases, can shield the alkylation event itself.

Figure 3.14 Structure of N-Boc-DSA, and N-Boc-CPI.

157 There would certainly appear to be a tentative relationship between increased side chain size and reduced alkylation activity. This is apparent in the observation that the alanine analogue appears to alkylate DNA more efficiently then the phenylalanine analogue, which in turn is better than the lysine and glutamic acid analogues. However the serine and β-alanine analogues both break this trend. The serine analogue has a larger side chain than alanine, but is as good as the ester control 42 at alkylating DNA. Again this could be the result of a positive effect specific to the presence of the hydroxyl group, and as mentioned previously, this will be discussed later. The β-alanine analogue has no side chain and yet performs as badly as the lysine and glutamic acid analogues. This is consistent with its apparent reduced activity in the MTS assay when compared to the alanine analogue. Clearly the size of the side chain could not be the only factor.

The apparent reduced alkylation efficiency of the β-alanine analogue compared to the alanine analogue may be a consequence of the greater flexibility of the linear structure of β-alanine, compared to the branched structure of alanine. For example this increased flexibility may incur a greater entropic cost to binding to the minor groove. This would also be true for the flexible side chains of the lysine and glutamic acid analogues. The greater entropic cost might be in part countered by the entropically favourable displacement of ordered water molecules known to be associated with the minor groove.^{191, 192} However, given the comparable size of the β-alanine and alanine analogues, the increased entropic penalty could be significant.

Figure 3.15 Schematic representation of a potential reason for the observed reduced alkylation efficiency between the alanine and β-alanine analogues.

158 Another potential negative consequence of the greater flexibility of the β-alanine structure can also be envisioned. In order to produce the greatest distance between the negatively charged C-terminal carboxyl group and the polyanionic phosphodiester backbone of DNA, the linear aliphatic chain of β-alanine, may bend to position the carboxyl group towards the inside floor of the minor groove. As a result this may produce a greater steric constraint on minor groove penetration of the alkylation subunit, than is produced by the branched less flexible structure of alanine (see figure 3.15). In such a configuration the β-alanine analogue appears to share more spatial homology with the glutamic acid analogue than the alanine analogue, and this is consistent with the observed alkylation activities.

As discussed, it is clear that the presence of the C-terminal amino acid structures are not altering the sequence selectivity of the alkylation event. However, it is possible that other non-covalent binding positions might be favoured thus reducing alkylation efficiency. For example, it was originally expected that the lysine analogue would be one of the best performing analogues, as it was hoped that the positively charged side chain might increase non-covalent binding affinity, by forming a favourable electrostatic interaction with the polyanionic phosphodiester backbone. This does not appear to be the case, and suggests that when the alkylation event occurs, the side chain of lysine may not be projecting out of the minor groove to associate with the phosphodiester backbone, but may lay across the floor of the minor groove, or be positioned towards the interior of the minor groove. Therefore it is possible that the lysine analogue prefers to bind with its side chain projecting out of the minor groove, but that in this binding orientation, the cyclopropane is no longer in a favourable position to alkylate the N3 of adenine. Similar such alternative binding orientations may exist for the other analogues.

It is most likely that the true reason for the reduced alkylation efficiency of the truncated amino acid-ducarmycin analogues (if real) is a complicated combination of different interplaying factors, which might include some of the ideas discussed above. The reduced activity also seen in the MTS assay, is likely a further more complicated interplay between the factors contributing to reduced DNA alkylation efficiency, coupled with factors contributing to reduced cell permeability.

The lysine extended analogue 44 containing the N-terminal methoxyindole subunit was also tested in the thermal cleavage assay. Consistent with the results of the MTS assay, the extended structure performed considerable better than the truncated N-acetyl analogues. Figure 3.16a, shows the DNA cleavage gel of 44, over a 100 fold concentration range (1 µM to 0.01 μM). Run in comparison on the same gel were the

N-159 acetyl C-terminal ester and acid compounds 42, and 43, at the previous 5 μM and 50 µM concentrations.

The first observation is the far superior DNA alkylation efficiency. Alkylation was observable down to at least the 0.01 μM concentration. The increased alkylation activity was further highlighted by altering the incubation temperature. The gel in figure 3.16a, shows cleavage after incubation at 37 ^oC as before. However, figure 3.16b, shows the cleavage gel of all the compounds when incubated at a concentration of 10 µM at only 25

oC. Under these conditions, only the extended analogue 44, shows any observable alkylation bands.

These results are entirely consistent with the reduced reactivity in thermal cleavage assays of truncated duocarmycin analogues such as N-Boc-DSA compared to the full natural product, which has be shown to alkylate DNA at lower incubation temperatures.¹⁸⁹ The trimethoxyindole subunit of the natural product, and the methoxyindole subunit of 44 likely improve DNA alkylation in the same manner. This is generally accepted to occur via two processes. Firstly, the methoxyindole subunit improves hydrophobic non-covalent minor groove binding affinity. Secondly, the increased linear length induces greater in situ activation of the cyclopropane, as described in chapter one.

It is likely that the improved activity of 44 compared with its truncated counterpart 39 in the MTS assay, is the result of both improved DNA alkylation efficiency, and superior cell permeability; both as a consequence of the methoxyindole subunit. However, as 44 clearly out performs the truncated C-terminal ester compound 42 in the thermal cleavage assay, but returned a higher IC₅₀ in the MTS assay, this compound is likely to still suffer from some reduction in cellular uptake, when compared to the C-terminal ester.

In addition to the improved alkylation activity, 44 also displays greater discrimination in alkylation sites when compared to the truncated compounds. This change in sequence selectivity is again consistent with what would be expected from the extended structure which is more similar to the full natural product.¹⁸⁹ The sequence selectivity of alkylation was quantified by measuring the intensity of the radioactive signal of each band. The results have been plotted as a proportion of the total cleaved DNA, against the sequence of the MS1 fragment in figure 3.17. Filled bars corresponded to cleavage by 44, and open bars to that of 42.

160 Figure 3.16 (a) DNA thermal cleavage gel of 44, 42, and 43 at various concentrations (μM).

Incubation at 37 ^oC. (b) DNA thermal cleavage gel of all the analogues at 10 µM. Incubation temperature 25 ^oC.

161 As discussed, while 42 demonstrates little selection other than the requirement for a 5’ A or T base neighbouring the alkylated A, 44 exhibits a clear preference for two sites within the MS1 sequence; one corresponding to 40 % of the total alkylation and the other to 26

%. The remaining 36 % of the total alkylation observed is spread relatively equally over six additional minor sites, but does range from 1 % to 13 %.

With the exception of the anomalous 1 % binding site, there would appear to be an absolute requirement for at least two 5’ A or T bases neighbouring the alkylated A. There is also a clear preference for longer AT rich sequences. The most favoured alkylation site (40 %) represents the longest AT rich run, comprising of four flanking 5’ A or T bases (5’-AATTA). In addition the second most favoured alkylation site (26 %) contains three 5’ A or T bases (5’-AATA). It would also appear that a flanking 5’ T base is favoured over A. This is apparent in the observation that in only two out of the eight sites alkylated by 44, did the first neighbouring 5’ base constitute an A. Furthermore, in the 13 % alkylation site, 44 has alkylated the A neighbouring the T base, when a longer 5’ AT rich sequence could have been achieved by alkylating the furthest 3’ A of the tetranucleotide AT rich sequence (5’-TTAA). Additionally, the only alkylation site not to contain more than one 5’ flanking A or T bases (the 1% site), happens to consist of a TA step (5’-TA).

These results are broadly in agreement with the sequence selectivity of duocarmycin SA, where in a different DNA fragment the major alkylation site was also found to constitute 5’-AATTA.¹⁸⁹ In line with duocarmycin SA the preference for longer AT rich sequences is most likely a consequence of the increased length of 44, compared to the truncated N-acetyl analogues. Here longer sequences are required to benefit from the increased

Figure 3.17 Intensity of each cleavage band, plotted as a proportion of total cleavage against the sequence of the MS1 DNA fragment. Open bars = 42. Filled bars = 44.

162 hydrophobic binding affinity provided by the narrower groove topology of AT rich regions.

In addition longer G free sequences are required to prevent the steric inhibition of groove penetration, by this base’s minor groove occupying amine.

3.13 Potential explanation for the possible superior activity of the