Outside-in and inside-out approaches to drug discovery for genetic diseases. (A) Outside-in screens start with a small-molecule screen vs. a model organism model of the genetic disease, followed by target identification (ID), validation, building an expanded library of small molecules based on structure activity relationships (SAR), and declaring a candidate drug for in vitro/in vivo pharmacokinetics (pharm)/absorption/distribution/metabolism/excretion (ADME)/ toxicology (tox) studies subsequent to a first-in-human trial. (B) Inside-out screens start with inhibition (normally) of a drug target being hypothesized to be able to ameliorate the genetic disease under study, followed by target validation in model organisms, a high-throughput screen for small molecules that inhibit the drug target, building an expanded library of small molecules based on SAR, determining the efficacy of lead compounds in model organisms, declaration of a candidate drug for in vivo pharm/ADME/tox, and a subsequent first-in-human trial. For both approaches, if the drug screen results in a known drug being identified that has efficacy in a model organism mimic of the genetic disease, then studies may be able to proceed directly to a first-in-human trial for disease treatment.

Application of yeast for drug discovery for genetic diseases. (A) A small molecule (drug) is screened vs. the ∼5000 separate nonessential yeast gene deletion set and is compared to the known yeast deletion array synthetic lethal genetic interactions. The yeast gene deletion(s) with a similar set of interactors to the small molecule can point to the pathway or gene/protein targeted by the small molecule. (B) HIPHOP (haploinsufficiency profiling or homozygous profiling) makes use of the diploid haploinsufficiency collection of all yeast genes. The entire collection is exposed to a small molecule and the culture is allowed to grow. Using bar-coding technology, the yeast strain(s) whose growth is decreased compared to a no drug control can be identified (ID). The small-molecule drug target should show decreased growth in a strain that is haploinsufficient for its target compared to the other strains. (C) Several applications of synthetic genetic array technology can be used to identify drug targets. The example illustrated works on the principle that inducible overexpression of a human open reading frame (ORF) in yeast results in an easily measurable phenotype (often growth). The yeast strain containing the human ORF (uninduced) can be mated to each of the ∼5000 nonessential yeast gene deletion strains and, through a series of strain selection steps, can result in the isolation of haploid yeast strains each containing the human ORF under control of an inducible promoter in each of the ∼5000 individual yeast gene deletion strains. Expression of the human protein can be induced, and yeast gene deletion strains that prevent its toxicity point to potential drug targets whose inhibition could prevent the toxicity of the human protein.

Applications of the zebrafish model in the study of inherited and acquired human genetic diseases. (A) Zebrafish and humans share a high degree of genetic conservation, including chromosome synteny and protein-coding genes. Molecular networks are also conserved, permitting use of zebrafish for investigating downstream pathways implicated in both inherited human diseases and somatic mutations like those found in cancer. Moreover, as a vertebrate model, the presence of analogous organs and conserved metabolism enable functional studies of the impact of genetic mutations on specific tissues and predictive readouts of therapeutic responses to drug administration. (B) The zebrafish is amenable to a number of genetic tools, including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing and transgenic technology, to introduce specific human mutations, including inserting oligonucleotides into the zebrafish genome. Other tools—including the facile introduction of plasmid DNA, BACs, and YACs; mRNA for transient gene overexpression studies and morpholinos for transient gene knockdown; and chemical mutagenesis—make the zebrafish a versatile model system for gene manipulation and phenotypic readouts. (C) Genetically modified zebrafish are easily surveyed using light and fluorescent microscopy, which can be enhanced with the availability of Lightsheet technology and confocal imaging. Whole-mount in situ hybridization provides tissue-specific data on RNA expression levels. Xenotransplantation of human cells, and in particular cancer cells, provides a transparent in vivo environment for real-time visualization of cell–microenvironment interactions. Embryos can be subjected to forward and reverse genetic screens. Automation of embryo sorting facilitates throughput and feasibility. (D) Translational outputs include preclinical therapeutic studies using compound libraries, dissection of molecular interactions in the context of a given genetic mutation, and novel gene discovery through phenotypic evidence of a causative mutation.