A Target Class Ligandability Evaluation of WD40 Repeat-Containing Proteins.

Target class-focused drug discovery has a strong track record in pharmaceutical research, yet public domain data indicate that many members of protein families remain unliganded. Here we present a systematic approach to scale up the discovery and characterization of small molecule ligands for the WD40 repeat (WDR) protein family. We developed a comprehensive suite of protocols for protein production, crystallography, and biophysical, biochemical, and cellular assays.

Identification of nonstandard macrocyclic peptide ligands through display screening.

Techniques facilitating the synthesis and screening of very high diversity nonstandard macrocyclic peptide libraries have led to such compounds receiving increasing attention as potential drug candidates. Specifically, approaches which allow the use of non-proteinogenic amino acids are proving to be particularly effective, since they expand the accessible chemical space of the starting library and thus allow the identification of compounds with structural similarity to known drugs. This review focuses on mRNA display screening platforms for drug discovery and their combined use with genetic code reprogramming to identify novel macrocyclic peptides with high affinities for disease-related targets of interest.

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing.

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding.

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole-Imidazole Polyamide seco-CBI Conjugates.

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon 12 mutation sequence.

Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate.

Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis.

Alteration of epigenetic program to recover memory and alleviate neurodegeneration: prospects of multi-target molecules.

Epigenetic chromatin remodeling and signalling pathways play an integral role in transcription dependent neurodegeneration and long-term potentiation (LTP), a cellular model associated with learning and memory. Pathological epigenetic modifications associated with neurological disorders are inherently flexible and can be reversed through pharmacological intervention. Small molecules are the favored drugs for clinicians, and in neurological disorders associated with complex cellular mechanisms, epigenetic and/or signalling pathway enzymes inhibiting small molecules have shown clinical prospects.

Sequence-specific DNA alkylation by tandem Py-Im polyamide conjugates.

Tandem N-methylpyrrole-N-methylimidazole (Py-Im) polyamides with good sequence-specific DNA-alkylating activities have been designed and synthesized. Three alkylating tandem Py-Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high-resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence.

Specific alkylation of human telomere repeat sequences by a tandem-hairpin motif of pyrrole-imidazole polyamides with indole-seco-CBI.

We designed and synthesized a tandem-hairpin motif of pyrrole (P)-imidazole (I) polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates (1) that targets the human telomere repeat sequence 5'-d(CCCTAA)n-3'. As a control, conjugate 2 (hairpin PI polyamide with seco-CBI), which also targets the human telomere repeat sequence, was synthesized. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) using 5' Texas Red-labeled 219-bp DNA fragments revealed the outstandingly high sequence selectivity of 1, with no mismatch alkylation.

Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively.

Sequence-specific DNA alkylation targeting for Kras codon 13 mutation by pyrrole-imidazole polyamide seco-CBI conjugates.

Hairpin N-methylpyrrole-N-methylimidazole polyamide seco-CBI conjugates 2-6 were designed for synthesis by Fmoc solid-phase synthesis, and their DNA-alkylating activities against the Kras codon 13 mutation were compared by high-resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5'-ACGTCACCA-3' (site 2), including minor 1 bp-mismatch alkylation against wild type 5'-ACGCCACCA-3' (site 3). Conjugate 6, which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5'-ACGTCACCA-3' (site 2).