Bradley J. Backes

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmins physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema

Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre‐clinical model of angioedema.

From Split‐Pool Libraries to Spatially Addressable Microarrays and Its Application to Functional Proteomic Profiling

Small molecules encoded with peptidonucleic acid (PNA) were used to probe protein function in a microarray format. The PNA tag served to encode the synthetic history of the small molecule and to positionally encode the identity of the small molecule by its location upon hybridization to an oligonucleotide microarray.

Substrate specificity of the human proteasome

BACKGROUND Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.

Solid support linker strategies

The selection of an appropriate linker is critical to the success of any strategy for the solid-phase synthesis of small molecule libraries. While the primary function of the linker is to covalently attach the initial substrate to the support, innovative strategies have emerged recently in which linkers fulfill important auxiliary roles. These include the cleavage of compounds into solution leaving no trace of the support attachment site, cleavage via cyclization, cleavage by introduction of additional diversity into the structure, and cleavage whereby portions of the compound are sequentially released into solution.

Structural and Functional Analysis of the Allosteric Inhibition of IRE1α with ATP-Competitive Ligands

The accumulation of unfolded proteins under endoplasmic reticulum (ER) stress leads to the activation of the multidomain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. We find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Further insight into how ATP-competitive inhibitors are able to divergently modulate the RNase domain through the kinase domain was gained by obtaining the first structure of apo human IRE1α in the RNase active back-to-back dimer conformation. Comparison of this structure with other existing structures of IRE1α and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1α oligomeric state and subsequent RNase domain activity.

Erratum: Erratum to “Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin”

On p. 188 of the February 2000 issue, “Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin” by Bradley J. Backes, Jennifer L. Harris, Francesco Leonetti, Charles S. Craik, and Jonathan A. Ellman, Figure 2 was incorrectly displayed.

Synthesis of C-11 modified mifepristone analog libraries

Substitution of the C-11 aniline of mifepristone can provide compounds with altered pharmacokinetic and pharmacodynamic (PK/PD) profiles that may find use for new indications. The development of new steroid intermediates and specialized library synthesis methods were required to enable the efficient preparation of structurally complex C-11 modified mifepristone analogs.