Advancing small-molecule-based chemical biology with next-generation sequencing technologies.

Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening.

Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of ß-alanine.

The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the ß-alanine that links SAHA to PIP, and led in turn to the design of ßß-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of ß-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove.

Identification of a small molecule that turns ON the pluripotency gene circuitry in human fibroblasts.

A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinical-friendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed Ì that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts.

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. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

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. Recently, small molecules with two or more functionalities, such as sequence-specific recognition and signalling pathways and/or enzyme modulation, have shown capabilities as efficient transcriptional activators. Here, we give a balanced overview of the key factors associated with memory recovery and neurodegeneration, available chemical tools for modulation and the demand to develop next-generation small molecules with multi-functional activities to treat such intricate, multi-gene associated neurological disorders.