Beyond traditional pharmacology: new tools and approaches

Abstract

Traditional pharmacology is defined as the science that deals with drugs and their actions. While small molecule drugs have clear advantages, there are many cases where they have proved to be ineffective, prone to unacceptable side effects, or where due to a particular disease aetiology they cannot possibly be effective. A dominant feature of the small molecule drugs is their single mindedness: they provide either continuous inhibition or continuous activation of the target. Because of that, these drugs tend to engage compensatory mechanisms leading to drug tolerance, drug resistance or, in some cases, sensitization and consequent loss of therapeutic efficacy over time and/or unwanted side effects. Here we discuss new and emerging therapeutic tools and approaches that have potential for treating the majority of disorders for which small molecules are either failing or cannot be developed. These new tools include biologics, such as recombinant hormones and antibodies, as well as approaches involving gene transfer (gene therapy and genome editing) and the introduction of specially designed self-replicating cells. It is clear that no single method is going to be a 'silver bullet', but collectively, these novel approaches hold promise for curing practically every disorder.

Figure 1

Therapeutic tools and approaches. Schematic illustration of currently used (small molecule drugs and biologics) and emerging novel therapeutic tools by category. Gene therapy includes the delivery of WT and modified genes, as well as constructs designed to reduce the expression of a particular gene (DNA sequences coding for shRNA and miRNA, as well as siRNA that are delivered directly). All constructs can be delivered using the most common virus-based, as well as non-viral, methods. Genome editing changes the actual DNA sequence in the genome. Three types of molecular tools were developed to achieve genome editing: zinc finger nucleases, TALENs, and the CRISPR-Cas system. Cell-based therapies involve the use of living cells for therapeutic purposes. The objective can be either to replace the cells that have degenerated or are missing, such as dopaminergic substantia nigra neurons in Parkinson's disease, or insulin-producing pancreatic beta cells in type I diabetes, or to introduce mammalian or bacterial cells performing a therapeutic task (e.g. producing a necessary compound).

Figure 2

Principles and applications of genome-editing techniques. (A) Genome-editing techniques utilize nucleases guided to specific sites by DNA-binding proteins that recognize specific DNA sequences or by RNA to cleave genomic DNA. ZFNs utilize nucleases such as Fok I coupled to zinc finger DNA-binding protein domains, which are used to create ZFNs. The TALEN-based approaches uses TALE domains linked to nucleases to create TALENs. Both systems are effective but require the design of new DNA-binding proteins for each DNA sequence to be targeted. Predicting the structure of zinc finger-binding protein domains specific for target genomic sequences remains challenging, and constructing TALENs is also demanding due to multiple repeat sequences. The newer system based on CRISPR utilizes the prokaryotic immune defense mechanisms. The main advantage of this technique is that the Cas9 nuclease is guided to the DNA locus by RNA, which is much easier to design and produce than a protein. (B) Nucleases introduce a double-stranded break in the target genomic sequence, which is repaired by the cell's DNA repair mechanism. The repair could be done via non-homologous end joining (NHEJ) creating a mismatch in the gene and thus inactivating it. Alternatively, the DNA break could be repaired using an endogenous or exogenous DNA template. In this case, a portion of a defective gene could be replaced via homology-directed repair (HDR) or a new gene could be inserted. (C) The CRISPR system could be employed to regulate the expression of endogenous genes. Deactivated Cas9, lacking nuclease activity, conjugated with a transcription activator could bring the activator to a specific gene locus. Alternatively, a transcription inhibitor could be delivered to a gene via nuclease-dead Cas9 that would block transcription initiation or elongation. For experimental purposes, deactivated Cas9 conjugated with fluorescent proteins such as GFP could be employed to visualize specific gene loci.

Figure 3

Phosphorylation-independent arrestin-1: expected compensation and unexpected side effects. (A) Mouse arrestin-1 was ‘pre-activated’ by triple alanine substitution of the hydrophobic residues in the C-tail (3A mutation; highlighted in magenta). (B) This mutation detaches the C-tail and dramatically enhances arrestin-1 binding to light-activated unphosphorylated rhodopsin (Rh*). (C) WT arrestin-1 cannot shut off rhodopsin signalling in the absence of rhodopsin kinase (RK−/−), so the time of half-recovery changes from ∼0.4 to ∼19 s. The expression of the arrestin-1-3A mutant instead of WT arrestin-1 in the absence of rhodopsin kinase (3A-50^{arr−/−rk−/−}) accelerates the recovery, reducing the time of half-recovery to ∼6 s (data from Song et al., 2009). (D) However, the 3A mutation also reduced the ability of arrestin-1 to oligomerize, greatly increasing the concentration of free monomer in the animals expressing the arrestin-1-3A mutant. This caused rapid photoreceptor degeneration, which is shown as a reduction in the outer nuclear layer (ONL) containing photoreceptor nuclei (data from Song et al., 2013). OS, outer segments, where rhodopsin and all components of the signalling cascade are localized; IS, inner segments, where photoreceptors rich in mitochondria are localized; ONL, outer nuclear layer, where the nuclei of photoreceptors reside; OPL, outer plexiform layer, where photoreceptor synapses are localized; INL, inner nuclear layer, where the post-photoreceptor neurons are localized. (E) Another source of problems is likely to be the release of the C-tail, which contains the AP2-binding site (shown as red oval in A). Arrestin-1 binds AP2 with ∼30-fold lower affinity than non-visual subtypes. Due to its very high expression in rods, even this low affinity matters: the association of full-length arrestin-1 with the constitutively active rhodopsin-K296E mutant causes photoreceptor death, whereas the replacement of the WT protein with arrestin-1 lacking the C-tail (this deletion pre-activates it similar to the 3A mutation) prevents photoreceptor loss (as reflected in the preservation of ONL thickness) and supports the functionality of photoreceptors expressing rhodopsin-K296E (data from Moaven et al., 2013).