According to the World Health Organisation (WHO) statistics (2014), infections are responsible for two to five of the top ten causes of death. In the remaining causes, infections can affect the outcome such as in cardiovascular or chronic obstructive airway diseases.
Antibiotics, used primarily to fight these infections, are one of the most important medical discoveries and their introduction has allowed for the treatment of various diseases once thought impossible. However, their extensive use and misuse has led to the worldwide spread of antibiotic resistant bacteria making it among the greatest global health challenges to be addressed. Livermore (2003) adds that some resistances are more widespread than others and multi-drug resistant strains are critical to the total accumulation of resistance examples of which include methicillin-resistant Staphylococcus aureus (MRSA).
A plethora of international agencies have developed public health initiatives to slow the progress of antibiotic resistance. Some of these initiatives advocate less antibacterial use and better use of antibiotics by educating physicians on antimicrobial therapy to encourage more appropriate prescription of antibiotics. Others include better infection control by increasing public awareness on the importance of compliance and hygiene as well as the development of new antibiotics.
Piddock (2011) summarises that despite the increasing need for new drugs with anti-microbial activity, antibiotic drug discovery and development has not seen significant progress in recent years. This could potentially be as a result of low return on investment by large pharmaceutical companies. This perceived low return on investment in the discovery of new antibiotics is due to the short courses and the current restrictions on their use.
Nevertheless, there is doubt as to whether reduced use or better prescription of antibiotics can substantially reverse growing resistance so overall, the way forward will be an improved balance between the increase in resistance and development of new antibiotics.
In order to match the rate of accumulation of resistance, an approach to speed up drug development is to examine new uses for approved existing drugs. This method, defined as ‘drug repurposing’ or ‘drug repositioning’ by Swamidass (2011) has become increasingly popular in recent times. Some examples of successfully repurposed drugs include Sildenafil; originally indicated for heart disease but now used as treatment for erectile dysfunction and thalidomide which is now indicated for multiple myeloma after being originally indicated for morning sickness but was found to have teratogenic effects.
Issa et al. (2013) describe the mainstay of drug repurposing efforts as the knowledge of drug chemical properties and this approach is based on the principle that similar molecules exhibit similar properties, bind similar protein targets and elicit similar biological effects.
Several strategies are practiced to identify potential drug leads for the purpose of repurposing approved drugs. These include lead discovery techniques such as high-throughput screening (HTS) and virtual screening (VS). The goal of HTS and VS according to Forli (2015) is to derive active compounds using experimental and computational approaches respectively. Stumpfe et al. (2012) agree and add that the ‘holy grail’ of the VS methods is its ability to detect hits which are structurally different from active compounds. For the purpose of this research, virtual screening will be discussed as a lead discovery technique and how it complements high throughput screening.
Virtual screening is becoming an increasingly popular method for identifying and selecting biologically active molecules as it makes more efficient use of time, resources, hit-rates and is less costly compared HTS (Kar and Roy, 2013). It is also able to explore a larger part of the drug-like chemistry space as it does not require physical availability of screening libraries.
In spite of these advantages, comparative studies suggest that VS and HTS are better utilised complementarily that competitively.
HTS involves screening a large library of small molecules or ‘hits’ against specific targets through ligand-docking based methods to identify ‘lead’ molecules which are them carried on for further studies.
In hit-to-lead identification, HTS and VS can be used complementarily in other ways to boost screening efforts. They can be used simultaneously in the same screening library (parallel screening) or sequentially. This integration of VS and HTS is an efficient approach for lead identification as it leads to the generation of libraries with more desirable chemical and biological properties.
A prerequisite for target based antibiotic discovery against resistant bacteria such as MRSA, is the identification of proteins necessary for growth and susceptible to inhibition by small molecules. Sequencing the genome of MRSA will aid in identifying essential genes and proteins for antibacterial development. Though this process can help to find many protein targets, these targets are not often abundant in sufficient quantities in the cell or accessible to small molecule inhibitors. Thus, new approaches have been developed to prioritise and select targets for drug discovery (Liu et al., 2004). Piddock (2011) agrees and adds that genetic experiments involving the inactivation or deletion of specific genes attenuate the bacteria thereby detecting a number of exploitative new targets that can be inhibited by antimicrobial action. The article gives further examples of approaches from academic research used to identify new antibacterial agents or augment existing ones. Some of these include exploitation of bacteriophages that target specific proteins, antisense inhibition of multi-drug transporter genes using licensed drugs and small-molecule inhibition of bacterial transcription factors.
A large number of potential drugs fail to advance to clinical and commercial use for various reasons. This makes them good candidates for repurposing. Approved drugs marketed commercially for a particular indication could also be repurposed based on its off-target effects. This method greatly reduces the cost of drug development in pre-clinical and phase 1 safety evaluation stages as these drugs may have passed the point of patent enquiry, failed to show efficacy in late stage clinical trials, stalled in development for commercial reasons or are being explored in new geographical markets (Sleigh and Barton, 2011). Drug repurposing serves to discover novel useful activities for these drugs with known biological activity by screening them against relevant disease targets. This research seeks to exploit the economic advantage of drug repurposing over the discovery of new chemical entities by screening a number of known drugs and assessing their inhibitory effects on protein targets derived from essential genes of MRSA.
According to McGregor et al. (2007), virtual screening approaches utilise target dependent and independent algorithms for hit-to-lead identification depending on whether the known small molecules are used as screening templates or the 3D structure of the target is utilised.
Target independent methods usually include a wider set of considerations as they are not dependent on knowledge of the target structure or their ligands. The aim of target independent methods is to screen for properties that classify a compound as ‘drug-like’. Some of these methods then include calculation of ADME/Toxicity (in vivo absorption, distribution, metabolism, excretion and toxicity) properties to eliminate clearly undesirable compounds. Another is to consider the chemical diversity of the compound to ascertain its biological activity which can lead to the discovery of ligands from new compound classes and targets with little known information.
Target dependent methods are sub-divided into those that are ligand-based or receptor (protein)-based. The approaches used to query compound libraries for potential active substances include QSAR, substructure analysis, pharmacophore analysis or any simpler method of measuring molecular similarity. It is only when the 3D structure of a protein target is known that docking methods can be employed.
This research will be utilising the target-based method because drug repurposing negates the need for target independent methods as the biological activity of the molecules have been previously ascertained. This method known as ‘docking’, does not require information from known ligands but utilises the key-lock principle of ligand –protein interactions. Hence, this method provides the opportunity to generate compounds that are truly novel.
Random screening of large compound libraries as in HTS is a major drug discovery method employed when there is little or no known information about the target. Despite advancements in technology which have increased the rate of screening of compounds in HTS, it is not always practicable. This may occur if the assay format cannot be scaled up to HTS format or if only a low-throughput assay is available. In such situations, the design of focused libraries where the selection of compounds is directed towards individual targets or classes of targets using various computational methods (Sotriffer, 2011).
For the purpose of this research, virtual screening will be employed for hit-to-lead identification from a number of small molecule drugs by docking their 3D models against protein targets derived from essential genes. The information derived from knowing the structure of these protein targets from MRSA genome sequencing coupled with knowledge of the structure of the drugs and their biological activity, will contribute to potential successful repurposing.
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