DESREGULACIÓN DE LAS CÉLULAS B POR EL VIH-1

6.2.1 Background information

While it is clear that clemastine 41 is an effective inhibitor of the Leishmania major IPCS, the mechanism by which this activity is achieved is unknown. Therefore, the precise action of clemastine-like inhibitors requires experimental verification. As X-ray crystallographic methods are not easily applicable to the membrane bound IPC synthase, other approaches must be employed. The use of photoaffinity probes, coupled with mass spectrometry (and MS/MS) analyses, have been reported in the literature for the identification and characterisation of protein targets.^262,263 In this, a protein and photoactive probe are incubated until sufficient binding is achieved. On irradiation, a highly reactive species, such as a nitrene or carbene, is generated which inserts into the protein to provide a cross-linked species (Scheme 6–2).

NBD-C6-ceramide

NBD-C6-IPC

LmjIPCS ScIPCS

Aureobasidin A (5 µM) − + − +

Subsequent proteolytic cleavage (e.g. using trypsin) yields fragments which can then be analysed using mass spectrometry. Comparing the output with that obtained from non-labelled protein enables the sites of probe-binding to be elucidated. Confirmation can be obtained by modifying these residues via site-directed mutagenesis to create engineered proteins which are resistant to the probe molecule.²⁶⁴

Scheme 6–2: Characterisation of a protein using a photolabelling-mass spectrometry experiment

A suitable probe molecule must fulfil certain criteria, such as chemical stability under enzymatic conditions, short irradiation period (to minimise potential damage to the protein target), a suitable wavelength for irradiation and a low propensity for non-specific labelling (i.e. irradiated species have a life span which enables efficient crosslinking).^263,265 Importantly, a synthesised probe must have comparable activity to the inhibitor it is based on. In addition, a photoaffinity probe may also bear other features such as fluorescent moieties, biotin residues or radioisotopic labels, to allow for facile identification and separation of labelled peptides.²⁶² This method could potentially be used with membrane-bound proteins, such as LmjIPCS, where a crosslinked probe may be used as a purification tag, to remove the enzyme from other membrane components prior to analysis.

The most commonly used photoreactive groups are benzophenones 159, aryl azides 160 and diazirines 161 (Scheme 6–3).²⁶⁵ Using clemastine 41 as a scaffold, it was viewed that introducing one of these functionalities onto an aryl ring would be relatively straightforward. A preliminary synthetic attempt focussed on the construction of aryl azide clemastine analogue 162, as it was assumed that the azide precursor 163 could be rapidly accessed.

Scheme 6–3: Example photoreactive groups

6.2.2 Synthesis of azide analogue 162

Following precedent by Andersen et al., it was viewed that the azide benzhydrol 163 could be accessed from the corresponding aryl bromide 164 (Scheme 6–4).²⁶⁶ 4-Bromobenzaldehyde 165 was reacted with freshly prepared phenyl Grignard reagent, furnishing the desired bromide precursor 164 in excellent yield, as evidenced by the presence of an IR-active O–H vibration at 3352 cm⁻¹.

Scheme 6–4: Synthesis of para-bromo benzhydrol 164

Next, aryl bromide 164 was reacted with sodium azide in the presence of a copper catalyst under microwave heating (Scheme 6–5).²⁶⁶ Infrared spectroscopic analysis of the resultant crude material revealed a characteristic azide vibration at 2120 cm⁻¹. Without purification, the crude azide 163 was coupled with homoprolinol (S)-57 to afford the desired ether 162, as confirmed by a 2D NMR HMBC experiment, which revealed coupling, between the benzylic (δH = 5.30 ppm) and methylene (δH = 3.53 – 3.43 ppm) positions.

Scheme 6–5: Synthesis of azide clemastine analogue 162

6.2.1 In vitro testing of azide analogue 162

The azide analogue 162 was tested against the L. major IPCS enzyme in a dose-response study (Figure 6–4). Azide 162 exhibited micromolar activity against the enzyme (IC50 = 3.66 µM, 95%

CI: 3.24 – 4.14 µM), at concentrations comparable to the clemastine analogue 78A (IC50 = 4.45 µM, 95% CI: 4.01 – 4.94 µM).

Figure 6–4: Biochemical activity of azide 162 against LmjIPCS. Nor-clemastine 78A is shown for comparison. Error bars portray 95% confidence interval.

Next, an assay against L. major promastigotes revealed the azide analogue 162 to have lower antileishmanial activity (ED50 = 2.85 µM, 95% CI: 2.28 – 3.56 µM) than nor-clemastine 78A by a factor of ~2 (ED50 = 1.69 µM, 95% CI: 1.23 – 2.32 µM) (Figure 6–5). The contribution of the azide moiety to this modest decrease in activity remains unclear.

Figure 6–5: Comparison of antileishmanial activity of azide 162 and clemastine analogue 78A. Error bars portray 95% confidence interval.

6.2.2 UV absorption measurement of azide 162

Once the inhibitory effect of azide 162 on L. major IPCS activity and promastigote growth was confirmed, the UV absorption profile of the potential photoaffinity probe was measured (Figure 6–6). Upon analysis of the obtained data, it was apparent that the absorption band of interest (235 – 275 nm) was similar to the ‘near-UV’ region (240 – 300 nm) generally observed in the UV spectra of proteins.^267,268 This is a common drawback of aryl azide probes,²⁶⁹ but may be circumvented by modifying substituents on the aromatic ring, thereby altering the photochemical properties of the probe.²⁷⁰ Measurement of the UV absorbance of LmjIPCS would allow for a direct comparison, however the presence of other membrane components would interfere, requiring the deconvolution of any obtained spectra. Furthermore, removal of the membrane lipids would risk unfolding the enzyme, which would furnish an unreliable UV absorption spectrum.²⁶⁸

Figure 6–6: UV absorption profile of azide 162 (blue line) at 25 µM in 50 mM phosphate buffer with 600 µM CHAPS. The green line represents blank buffer.

6.2.3 Conclusions

In conclusion, azide 162 was synthesised and found to maintain activity against the L. major IPCS enzyme and promastigote parasites, when compared to nor-clemastine 78A. However, an assessment of its potential as a photoaffinity probe revealed the lack of a suitable irradiation wavelength which would not be liable to interference from biological material.

Future work should focus on the synthesis of substituted aryl azides, which may influence UV absorption (Figure 6–7).²⁷⁰ Additionally, an exploration of other clemastine derivatives featuring the benzophenone and diazirine photoreactive groups may reveal a promising alternative compound series. Identification of a viable crosslinking moiety may then inform the syntheses of analogues bearing additional functionality, such as purification tags or fluorescent groups.

Figure 6–7: Other potential clemastine-based photoaffinity probes 0

0.5 1 1.5 2 2.5

190 200 210 220 230 240 250 260 270 280 290 300

Absorbance (AU)

Wavelength (nm)

UV absorption profile of azide analogue 162