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The second project that was carried out during this PhD thesis involved the investigation of the natural product symplostatin 4[103] that is also known by the name gallinamide A.[104] The interest in this natural product came from its molecular structure that consists of a prominent Michael acceptor system that has been recognized as a warhead for cysteine proteases and the rare C-terminal methoxy-methylpyrrolinone (mmp) residue as well as the interesting biological activities reported the compound. Consequently, symplostatin 4 seemed to be a natural product that could be a useful tool in chemical biology investigations, for example after conversion into an activity-based probe. Furthermore, the role of the mmp unit could be investigated by replacing this moiety with another amino acid.

To gain a quick access to symplostatin 4 (Sym4), an analog lacking the mmp moiety (Sym4mmp→Ala_{) and also derivatives of these substances that would function as probes in} ABPP, a highly convergent, fragment-based synthesis was developed (Fig. 85). To enable a flexible conversion of Sym4 and Sym4mmp→Ala _{into suitable probes for ABPP, alkyne-modified} versions of the two molecules were generated (≡Sym4 and ≡Sym4mmp→Ala_{). They were then} converted either into activity-based probes by reaction with azide-modified tags by a 1,3- dipolar Huisgen cycloaddition (click reaction) or used directly in a labeling and modified by a click reaction after the labeling. For the highest possible flexibility, rhodamine-, biotin- and trifunctional versions of both molecules were also generated (Fig. 86).

Figure 85: Convergent synthesis of symplostatin 4 and analogs. Reagents and conditions: (a) N,O-

dimethylhydroxylamine hydrochloride, Et3N, DCC, CH2Cl2, rt, 1 h; (b) LiAlH4, Et2O, 0 °C, 20 min; (c)

Ph3P=CHCOOMe, CH2Cl2, rt, o/n (59%, 3 steps); (d) LiOH, THF/MeOH/H2O (1.67:1:0.67), rt, 5 h; (e) H-Ala-OMe,

HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, o/n (46%, 2 steps); (f) LiOH, THF/MeOH/H2O (1.67:1:0.67), rt, 5 h; (g) 2,2-

dimethyl-1,3-dioxane-4,6-dione, DMAP, EDC*HCl, CH2Cl2, -10 °C to rt, o/n; (h) EtOAc, reflux, 1 h; (i) Ph3P,

MeOH, DEAD, THF, rt, o/n (90%, 4 steps); (j) TFA/CH2Cl2 (1:1), rt, 3 h; (k) Boc-Leu-OH, HOBt, HBTU, DIEA, CH3CN,

0 °C to rt, o/n (85%, 2 steps); (l) TFA/CH2Cl2 (1:1), rt, 2 h; (m) Boc-Leu-OH, HOBt, HBTU, DIEA, CH3CN, 0 °C to rt,

o/n (99%, 2 steps); (n) SOCl2 (2 M in CH2Cl2), MeOH, 0 °C to rt, o/n; (o) Boc-NMeIle-OH, DMAP, DCC, CH2Cl2, 0 °C

to rt, o/n (55%, 2 steps); (p) TFA/CH2Cl2, rt, 2 h; (q) CH3I, DIEA, DMF, rt, 2 h (38%, 2 steps); (r) propargyl bromide

(80% wt in toluene), DIEA, CH3CN, 80 °C, o/n (63%, 2 steps); (s) LiOH, THF/MeOH/H2O (1.67:1:0.67), rt, 5 h; (t)

TFA/CH2Cl2 (1:1), rt, 2 h; (u) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, 5 h (91% after silica gel chromatography, 51%

after additional HPLC, 3 steps); (v) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, o/n (39% after HPLC, 3 steps); (w)

HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, o/n (82% after silica gel chromatography, 64% after additional HPLC, 3

steps); (x) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, 5 h (73% after silica gel chromatography, 53% after additional

Figure 86: Synthesis of the differently modified probes for ABPP. Reagents and conditions: (b) Tag-N3, CuSO4

(aq. solution), TBTA (100 mM in DMSO), TCEP (100 mM in H2O), H2O, rt, reaction monitoring by LC-MS,

chromatographic work-up.

During a placement in the group of Dr. Renier van der Hoorn (MPI for Plant Breeding research, Cologne), symplostatin 4 and its derivatives were investigated for their labeling of the Arabidopsis thaliana proteome. In preliminary labeling experiments the optimal conditions for the in vivo labeling were established; additionally, some in vivo infiltration experiments were carried out, yet without remarkable results.

The target identification for Sym4 and Sym4mmp→Ala _{was then carried out by Dr. Farnusch} Kaschani, who was able to identify the papain-like cysteine proteases RD21A and RD21B as the major targets of Sym4 and Sym4mmp→Ala in A. thaliana by in vivo labeling of A. thaliana cells. RD21 has been assigned a role in the plant immune system.[150] Of note, a difference in the labeling pattern of Sym4 and Sym4mmp→Ala was not observed in these experiments (Fig. 87).

Figure 87: Fluorescence scan of the SDS-PAGE-resolved enzymes labeled in vivo by ≡Sym4 and ≡Sym4mmp→Ala_. The labeling was carried out in vivo, followed by an in vitro click reaction with Tri-N3 and affinity purification of

the labeled enzymes on agarose gel. After the resolution of the labeled enzymes, the individual protein bands were excised from the gel, digested with trypsin and analyzed by nanoLC-MS/MS. (work by F. Kaschani)

Having successfully identified papain-like cysteine proteases as the targets of symplostatin 4 in plant cells, the investigation of the antimalarial effect of symplostatin 4[104] was initiated under the hypothesis that the antimalarial activity might stem from an inhibition of the parasital falcipains. This project was carried out in collaboration with the group of Prof. Dr. Matthew Bogyo (Department of Pathology, Stanford University, CA); all experiments were carried out by Dr. Edgar Deu Sandoval.

In a first, cell-based assay the effect of Sym4 and Sym4mmp→Ala _{on malaria parasites in the} erythrocytic cycle became apparent: cells treated with 0.1 μM Sym4 showed a distinct

swollen food vacuole phenotype which is related to a disturbance of the proteases involved in the early stage hemoglobin digestion.[50,53,151] _{In contrast to this, the cells had to be}

treated with a 100-fold higher concentration of Sym4mmp→Ala _{to observe the same effect,} indicating a clear difference between the two analogs that might stem from either a decreased uptake of Sym4mmp→Ala or a less specific binding to the target enzyme(s) (Fig. 88, A). Importantly, at no applied concentration, a lysis of the red blood cells could be observed.

After having studied the direct phenotypic effect of symplostatin 4 on the living parasite, the overall effect on parasite replication was investigated. This allows to evaluate if the effect

observed at an early stage of the parasite life cycle has an influence on the total development and replication (i. e. the progression of the disease). Hence, cell cultures at 2% parasitemia were treated with different concentrations of the inhibitors for 75 h and after this time, the parasitemia was determined by flow cytometry (Fig. 88, B).[152] Again, a clear difference between Sym4 and its analog Sym4mmp→Ala became apparent, thereby confirming the results of the previous phenotype assay.

Since the results from the cell-based assays already indicated strongly that one or more of the parasite’s food vacuole cysteine proteases, i. e. falcipain 2, falcipain 2’ and falcipain 3 (FP-2, FP-2’, FP-3)[50] as the targets of Sym4, a focus was set on verifying this hypothesis by activity-based profiling experiments. Firstly, Rh-Sym4 and Rh-Sym4mmp→Ala were tested for their labeling of intact parasites. Indeed, two strong bands in the 28 kDa region appeared for

Rh-Sym4, while for Rh-Sym4mmp→Ala only a very weak labeling in this region could be detected. The bands were sensitive to a pre-incubation with Sym4 and ≡Sym4, indicating that the labeling is not an artifact caused by the rhodamine tag (Fig. 88, C). This labeling in the 28 kDa region was already a strong indicator for the inhibition of FP-2, FP-2’ _{and FP-3}

(referred to as FP-2/3) that are known to migrate in the 28 kDa region.[153,154] _{A competitive}

ABPP experiment with Cy5-labeled DCG-04 and the probes Sym4 and Sym4mmp→Ala _was therefore carried out to test if Sym4 and Sym4mmp→Ala _{indeed labeled FP-2/3.}

Sym4 was revealed to be a nanomolar inhibitor (1.5 nM) of FP-2/3; in addition, FP-1 was also

inhibited at higher concentrations (1.6 μM). The aminopeptidase DPAP-1 was not inhibited

(Fig. 88, D). Sym4mmp→Ala _{on the other hand inhibited FP-2/3 only weakly (25 μM); an} inhibition of FP-1 or DPAP-1 could not be detected at all.

For the potent FP-2/3 inhibitor Sym4 the parameters of inhibition were then further investigated. A substrate turnover experiment with recombinant FP-2 and FP-3 revealed that

Sym4 is an irreversible inhibitor of FP-2 and FP-3 and that it is approximately 10-fold more

potent against FP-2 than it is against FP-3. In summary, the assays revealed Sym4 as one of the most potent FP-2/3 inhibitors reported to date.[155] The determination of the IC50 values

for the different PLCPs revealed that Sym4 preferentially inhibits FP-2 over FP-3 and FP-1. Indeed, all falcipains are inhibited in the sub-micromolar range (Fig. 88, E). A summary of all

Table 6: Summarized antimalarial activities of Sym4 and Sym4 .

Sym4 Sym4mmp→Ala food vacuole

defect observed at 0.1 μM 10 μM

EC50

(from replication assay) 0.7 ± 0.2 μM 27 ± 7 μM

labeling of FP-2/3 in intact parasites at 10 μM strong weak

FP-2/3 inhibition starting from (comp. assay) 1.5 nM 25 μM

FP-1 inhibition starting from (comp. assay) 1.6 μM not observed

ki(FP-2) / M-1s-1 58600 ± 1400 not determined

ki(FP-2) / M-1s-1 7030 ± 250 not determined IC50(FP-2) 8.5 ± 1.3 nM not determined

IC50(FP-3) 22 ± 8 nM not determined

IC50(FP-1) 140 ± 23 nM not determined

Figure 88: Summarized results of the investigation of the antimalarial activity of Sym4. A: food vacuole

phenotype caused by Sym4 treatment of P. falciparum D10 at ring stage; B: effect of Sym4 treatment on replication of P. falciparum; C-E: ABPP experiments with Sym4 and ABPP analogs: labeling of intact parasite cells (C), competitive ABPP with Cy5-DCG-04 (D), competitive ABPP with Cy5-DCG-04 in order to determine IC50

In summary, the overall antimalarial activity of Sym4 most likely results from a combined inhibition of FP-2 and FP-3 (and to a lesser extent FP-1). The food vacuole defect observed at an early stage of the parasital development is most likely caused by the inhibition of FP-2; the decreased replication (i. e. killing of the parasite) is in contrast most likely caused by the inhibition of FP-3 that has been demonstrated to be the determining factor for parasite survival.[52]

In conclusion, the natural product symplostatin 4 has been studied in two different organisms. The targets of symplostatin 4 in A. thaliana have been identified to be the PLCPs RD21A and RD21B. For the labeling in the model plant, no difference in the labeling pattern between Sym4 and its analog lacking the putatively important mmp unit was observed. The antimalarial activity of symplostatin 4 was studied extensively, revealing Sym4 to be a highly potent inhibitor of the plasmodial falcipains. Here, a significant difference between

Sym4 and Sym4mmp→Ala _{was observed. Sym4 may in the future serve as a lead structure for} the development of new antimalarial drugs inhibiting the parasite’s falcipains; the different ABPP probes may furthermore be used for labeling experiments and investigation of different proteomes.