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Fig. 2. Synthesized -chloromethyl ketone peptides.substrate Ac-RRLL-MCA. The inhibitor peptides (RRLL-cmk derived peptides I-IV andDec-RRLL-cmk) stock solutions were prepared in DMSO at 10 mM peptide concentration.The solutions containing vv:hSKI-1-BTMD or vv:WT and inhibitor peptides or DMSOwere pre-incubated for 20 min at 37°C prior to initiation of the reaction by addition of thefluorogenic substrate Ac-RRLL-MCA. Enzymatic activity measurements were performedby monitoring the liberated AMC group with a SpectraMax Gemini EM microplatespectrofluorometer (Molecular Devices) ( ex =360 nm; em=470 nm with a cutoff filter setat 435 nm). For each inhibitor, the background activity measured in the presence of vv:WTwas subtracted from the enzymatic activity measured in the presence of vv:hSKI-1-BTMD.Results and DiscussionAny given peptide sequence can be synthesized as chloromethyl ketone (with availableamino acid chloromethyl ketone derivative) in the described way. As an example, wesynthesized several SKI-1 inhibitors and demonstrated their inhibitory potency andvalidation of used procedure (Figure 3).Fig. 3. The RRLL-cmk derivedpeptides I-IV and Dec-RRLLcmkshow similar inhibitory acti-100vity of the in vitro processing ofAc-RRLL-MCA by SKI-1. Inhibitorpeptides (20 mM) or DMSO80(control) and hSKI-1 were60preincubated for 20 min at 37°Cbefore initiation of the reactionby addition of 200 mM fluorogenicsubstrate, Ac-RRLL-MCA.40hSKI-1 activity is expressed as %20of the activity measured in thepresence of DMSO control(averages, n=2, ± S.D.).hSKI-1 Activity (% DMSO Control)0DMSODec-RRLL-cmkAc-RRLL-cmk (I)Ac-YRRLL-cmk (II)C11-YRRLL-cmk (III)Dec-YRRLL-cmk (IV)AcknowledgmentsThis work is supported by research grants to RD and NGS from the Canadian Institutes of HealthResearch (CIHR) and the Ministère du Développement Économique, de l'Innovation et del'Exportation (MDEIE) du Québec.References1. Kwon, Y., Welsh, K., Mitchell, A.R., Camarero, J.A. Organic Letters 6(21), 3801-3804 (2004).81