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Table 1. Inhibitory effect of CARD-derived synthetic peptides in apoptosome reconstitutionin-vitro assaysCARDproteinderivedHelix. a Peptide b Sequence IC 50 (µM) c IC 50 (µM) d2.63 YIMDHMISDG 90 66Apaf-13.65 FLTISEEEKV 102 841.66 LRRCRLRLVE 68 108PC94.67 RDQARQLII 81 (-)2.75 TQCLVDNLLKND (-) (-)Nod-13.76 YFSAEDAEIVCA (-) (nd)Nalp-1 3.78 VVLDKLHGQ (-) (-)a Represent the helix in the corresponding CARD domain. b Peptide code assigned.c Determined as Caspase-9 activity inhibition in apoptosome reconstitution withrecombinant proteins. d Determined as Caspase-3 activity inhibition in cell-free extracts;nd: not determinedIn both assays (Figure 1A and B) the peptides 2.63 and 3.65 derived from Apaf-1 CARDwere the most active as inhibitors, the IC 50 was around 60-100 uM (Table 1). The peptidesderived from Nod-1 and Nalp-1 did not present activity, confirming the specificity andselectivity from apoptosome derived peptides. Finally, at equimolar concentration thepeptides derived from CARD Apaf-1 presented synergic effect and inhibit at 100% theapoptosome in opposition to the lack of contribution of peptides derived from CARD-PC9(Figure 1C).The designed mimetic peptides presented inhibitory activity at moderate but selectivelevel and may represent the first generation of specific peptide apoptosome inhibitors.AcknowledgmentsY. P.-R. was supported by a postdoctoral fellowship from National Autonomous University of Mexicoand Consejo Superior de Investigaciones Científicas de España (UNAM-CSIC). The project wassupported by MICINN BIO2007-60066 and Generalitat Valenciana PROMETEO/2010/005.References1. Qin, H., Srinivasula, S.M., Wu, G., Fernandes-Alnemri, T., Alnemri E.S., Shi, Y. Nature 399, 549-557 (1999).2. Inohara, N., Nuñez, G. Nat. Rev. Immunol. 3, 371-382 (2003).3. Proell, M., Riedl, S., Fritz, J.H., Rojas, A. M., Schwarzenbacher, R. Plos one 3, e2119 (2008).4. Riedl, S.J., Li, W., Chao, Y., Schwarzenbacher, R.S., Shi, Y. Nature 434, 926-933 (2005).553