Compound 1541 and its analog (1541B) formed a unique cluster in this analysis

Compound 1541 and its analog (1541B) formed a unique cluster in this analysis. different views. (d) Chemi-fibrils induce cell death, but the monomers do not. 1541 or STS were added into a dialysis chamber which was placed into a dish of K562 cells. Cell viability after 24 hours was measured. (e) The non-ionic detergent Tween-80 disrupts 1541 cell-death activity, but has no effect on staurosporine (STS). Cell viability was monitored using CellTiter-Glo (raw luminescence unit). The data represent mean values s.d. (n=3). Here, using various biochemical and biophysical methods, we show that the chemi-fibrils, and not the free soluble small molecule, induce cell death in mammalian culture. We employed diverse methods including shRNA screens6-8, CDK-IN-2 chemical genetic approaches9, N-terminomics to identify proteases involvement10-12, modulatory profiling to help classify their cellular mechanism13, and cell biology tools to show how these enter cells and induce cell death. Remarkably, the chemi-fibrils enter through the endocytic pathway and traffic to lysosomes leading to activation of intracellular proteases, including caspases. We believe these synthetic chemi-fibrils may CDK-IN-2 provide important insights into how extracellular fibrillar structures can induce cell death. Results Structural characterization of 1541 Individual molecules of compound 1541 rapidly self-assembles into well-ordered nanofibrils as observed by electron microscopy4 (Fig. 1b). We wished to understand the intermolecular packing of 1541 because unlike protein forming fibrils, 1541 contain very little opportunity for hydrogen bonding. We determined the X-ray structure of 1541 at atomic resolution to reveal the intermolecular interactions between the small molecules, as shown in Figure 1c; crystal data and structure refinement can be found in the Supplementary Results and Supplementary Figure 1. The small molecules are strictly planar and stack on each other with a separation of 0.34 nm. However, each small molecule is shifted by 0.64 nm so that there is no perpendicular ring stacking. Considering that each 1541 molecule is 1.5 nm wide, and that each individual fibril is as thin as 2.6 nm, as observed by transmission electron microscopy (TEM), it is conceivable that the fibrils may be composed of only two to three 1541 molecules in width. Fiber diffraction studies will be needed to confirm that this packing arrangement is preserved in the chemi-fibrils, as well as to define the fiber axis. Nonetheless, these data show tight packing can be achieved in these chemi-fibrils forming molecules without intricate hydrogen bonding networks typical of proteinaceous fibrils14. Cell death is induced by the chemi-fibrils not monomers We have previously shown by TEM and dynamic light scattering (DLS) that the chemi-fibrils of 1541 form within the mixing time when added from DMSO to neutral buffers. The chemi-fibrils also form immediately when transferred from DMSO to cell culture media (Supplementary Fig. 2). Interestingly, the threshold concentration for formation of 1541 chemi-fibrils in cell culture (2 M) observed by DLS, matches the approximate EC50 for cell death induced by mammalian cells3. We have shown that once the chemi-fibrils have formed they are apparently kinetically trapped. For example, when a dialysis chamber is placed in a buffer containing 1541 chemi-fibrils, we cannot detect 1541 inside the chamber over a 12 hour period at 37C (Supplementary Fig. 3). Furthermore, 1541 will not activate procaspase-3 when the two are separated by a dialysis membrane4. Here, we conduct an analogous experiment to determine if 1541 can induce cell death when partitioned by a dialysis membrane. We utilized the immortalized myelogenous leukemia line K562, which is commonly used in cell death studies. Also, K562 cells conveniently grows readily in suspension and performs well in pooled shRNA screens7. As with other cell lines we CDK-IN-2 have tested, K562 cells are highly sensitive to 1541-induced cell death as monitored by drop in ATP levels and caspase activation (Supplementary Fig. 4). However, when K562 cells are exposed to 1541 sequestered in a dialysis bag (M.W. cutoff of 3.5 kDa) the cells do not undergo cell death over a 48-hour period (Fig. 1d and Supplementary Fig. 5). In contrast, staurosporine (STS; M.W. 466 Da), a promiscuous protein kinase inhibitor that induces apoptosis in a variety of cell types and that does not form aggregates, causes rapid cell death when cells are exposed directly or isolated behind the dialysis membrane. The same results occurred when cells were placed inside the dialysis bag and the small molecules outside (Supplementary Fig. 6)..2c); based on their protecting p-values, Rab1A, Rab2A and V-ATPase were rated #1, #2, and #4, respectively. (STS). Cell viability was monitored using CellTiter-Glo (uncooked luminescence unit). The data represent mean ideals s.d. (n=3). Here, using numerous biochemical and biophysical methods, we show the chemi-fibrils, and not the free soluble small molecule, induce cell death CDK-IN-2 in mammalian tradition. We employed varied methods including shRNA screens6-8, chemical genetic methods9, N-terminomics to identify proteases involvement10-12, modulatory profiling to help classify their cellular mechanism13, and cell biology tools to show how these enter cells and induce cell death. Amazingly, the chemi-fibrils enter through the endocytic pathway and traffic to lysosomes leading to activation of intracellular proteases, including caspases. We believe these synthetic chemi-fibrils may provide important insights into how extracellular fibrillar constructions can induce cell death. Results Structural characterization of 1541 Individual molecules of compound 1541 rapidly self-assembles into well-ordered nanofibrils as observed by electron microscopy4 (Fig. 1b). We wished to understand the intermolecular packing of 1541 because unlike protein forming fibrils, 1541 consist of very little chance for hydrogen bonding. We identified the X-ray structure of 1541 at atomic resolution to reveal the intermolecular relationships between the small molecules, as demonstrated in Number 1c; crystal data and structure refinement can be found in the Supplementary Results and Supplementary Number 1. The small molecules are purely planar and stack on each other having a separation of 0.34 nm. However, each small molecule is definitely shifted by 0.64 nm so that there is no perpendicular ring stacking. Considering that each 1541 molecule is definitely 1.5 nm wide, and that every individual fibril is as thin as 2.6 nm, as observed by transmission electron microscopy (TEM), it is conceivable the fibrils may be composed of only two CDK-IN-2 to three 1541 molecules in width. Fiber diffraction studies will be needed to confirm that this packing arrangement is maintained in the chemi-fibrils, as well as to define the dietary fiber axis. Nonetheless, these data display tight packing can be achieved in these chemi-fibrils forming molecules without complex hydrogen bonding networks standard of proteinaceous fibrils14. Cell death is induced from the chemi-fibrils not monomers We have previously demonstrated by TEM and dynamic light scattering (DLS) the chemi-fibrils of 1541 form within the combining time when added from DMSO to neutral buffers. The chemi-fibrils also form immediately when transferred from DMSO to cell tradition press (Supplementary Fig. 2). Interestingly, the threshold concentration for formation of 1541 chemi-fibrils in cell tradition (2 M) observed by DLS, matches the approximate EC50 for cell death induced by mammalian cells3. We have shown that once the chemi-fibrils have formed they may be apparently kinetically caught. For example, when a dialysis chamber is placed inside a buffer comprising 1541 chemi-fibrils, we cannot detect 1541 inside the chamber over a 12 hour period at 37C (Supplementary Fig. 3). Furthermore, 1541 will not activate procaspase-3 when the two are separated by a dialysis membrane4. Here, we conduct an analogous experiment to determine if 1541 can induce cell death when partitioned by a dialysis membrane. We utilized the immortalized myelogenous leukemia collection K562, which is commonly used in cell death studies. Also, K562 cells conveniently grows readily in suspension and performs well in pooled shRNA screens7. As with additional cell lines we have tested, K562 cells are highly sensitive to 1541-induced cell death as monitored by drop in ATP levels and caspase activation (Supplementary Fig. 4). However, when K562 cells are exposed to 1541 sequestered inside a dialysis bag (M.W. cutoff of 3.5 kDa) the cells do not undergo cell death over a 48-hour period (Fig. 1d and Supplementary Fig. 5). In contrast, staurosporine (STS; M.W. 466 Da), a promiscuous protein kinase inhibitor that induces apoptosis in a variety of cell types and that does not form aggregates, causes quick cell death when cells are revealed directly or isolated behind the dialysis membrane. The same results occurred when cells were placed inside the Mbp dialysis bag and the small molecules outside (Supplementary Fig. 6). One method to perturb small molecule aggregators is to use small amounts of non-ionic detergent in cell tradition15. Specifically, Tween-80 is able to dissolve small molecule aggregators and offers negligible toxicity in cell tradition when dosed less than 0.1% (Supplementary Fig. 7)15. We find that Tween-80 protects cells inside a dose dependent manner from 1541 induced cell death (Fig. 1e, remaining), but does not guard cells from killing.