Fragment‐based lead generation has proven to be an effective means of identifying high‐quality lead compounds for drug discovery programs. However, the fragment screening sets often used are principally comprised of sp2‐rich aromatic compounds, which limits the structural (and hence biological) diversity of the library. Herein, we describe strategies for the synthesis of a series of partially saturated bicyclic heteroaromatic scaffolds with enhanced sp3 character. Subsequent derivatization led to a fragment collection featuring regio‐ and stereo‐controlled introduction of substituents on the saturated ring system, often with formation of new stereocenters.

We describe a MALDI-TOF mass-spectrometry-based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALSG), CaMKII substrate (KRQQSFDLF), erktide (ATGPLSPGPFGRR), abltide (EAIYAAPFAKKK), srctide (AEEEIYGEFEAKKKK), neurogranin (AAAKIQASFRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMSITA). There are two fundamental points on which the proposed method is based to improve the mass-spectrometric response: 1) mass tag technology by N-derivatization through stable isotope labeling and 2) C-terminal conjugation with tryptophanylarginine (WR). It was suggested that C-terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5-13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac-EAIYAAPFAKKKWR-NH(2)), that WR conjugation at the C-terminus in combination with stable-isotope labeling at the N-terminus allowed the quantitative assay of recombinant c-Abl kinase in the presence of adenosine 5'-triphosphate (ATP; K(M,ATP)=18.6 microM and V(max)=642 pmol min(-1) microg(-1)). The present protocol made a simple and reliable inhibition assay of recombinant c-Abl kinase by imatinib possible (IC(50(recombinant))=291 nM; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c-Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC(50(active form))=1049 nM and IC(50(inactive form))=54 nM). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer-cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple-kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high-throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope-free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.

Recent technological innovations have led to the development of methods for the rapid identification of high-affinity macrocyclic peptides for a wide range of targets; however, it is still challenging to achieve the desired activity and membrane permeability at the same time. Here, we propose a novel small molecule lead discovery strategy, ″Peptide-to-Small Molecule″, which is a combination of rapid identification of high-affinity macrocyclic peptides via peptide display screening followed by pharmacophore-guided de novo design of small molecules, and demonstrate the applicability using nicotinamide N-methyltransferase (NNMT) as a target. Affinity selection by peptide display technology identified macrocyclic peptide 1 that exhibited good enzymatic inhibitory activity but no cell-based activity. Thereafter, a peptide pharmacophore-guided de novo design and further structure-based optimization resulted in highly potent and cell-active small molecule 14 (cell-free IC50 = 0.0011 μM, cell-based IC50 = 0.40 μM), indicating that this strategy could be a new option for drug discovery.