High-Throughput Screening (HTS) is one of the main sources of leads for drug discovery; the process involves evaluating a large diverse sample collection in an effort to identify compounds that possess a desired activity. In practice this resembles looking for a needle in a haystack since the vast majority of compounds in the sample collection will be devoid of the desired activity. The products of this activity are referred to as “Hits”, once the activity has been confirmed to be appropriate with respect to potency/selectivity, the chemical structure and purity established, and preliminary ADME completed, these hits are often then described as “Leads”. In reality advances in robotics have resulted in the actual screening being relatively rapid whilst the subsequent follow-up is often the slow (and expensive) step, thus it is essential all “Hits” are genuine and have a high probability to become acceptable “Leads”.
Factors effecting Screening Collection design
Physicochemical Properties
The lack of productivity in the Pharmaceutical Industry lead to a re-evaluation of HTS initially resulting efforts by companies to dramatically increase the size of the screening collection by employing combinatorial chemistry to prepare vast libraries of compounds. However this effort was largely unsuccessful and merely served to increase the size of the haystack rather than the number of needles. The seminal work by Chris Lipinski (Advanced Drug Delivery Reviews 46 (2001) 3 – 26) highlighted the importance of physicochemical properties in drug design leading to the “Rule of Five”. This states “In the discovery setting ‘the rule of 5’ predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5.” Since then a number of other studies have made similar observations that attrition in the drug discovery process is linked to physicochemical properties of the candidate compounds.
Most recently a study of preclinical toxicity (Bioorganic & Medicinal Chemistry Letters 18 (2008) 4872–4875), found, across a wide range of chemical space, a greatly increased likelihood of toxicity was found for less polar, more lipophilic compounds. This results are perhaps not surprising when one considers the structure-activity relationships (SAR) of a variety of potential liabilities:-
• HERG, large lipophilic active site
• Partition into liver enhanced by increased lipophilicity
• CYP3A and CYP2D6 inhibition increased by lipophilicity
• CP450 Induction, PXR binding site is large and hydrophobic
• Plasma Protein Binding, affinity increases with logD
With this in mind there have been several studies evaluating the changes in molecular properties in going from the initial lead to the drug candidate (J. Chem. Inf. Comput. Sci. 2001, 41, 1308-1315) leading to the following observations.
“The following differences were observed between the medians of drugs and leads: ∆MW = 69; ∆CMR = 1.8; ∆RNG = ∆HAC =1; ∆RTB = 2; ∆CLogP = 0.43; ∆LogD74 = 0.97; ∆HDO = 0; ∆DFPS = 0.15; ∆PPFS = 0.12. Lead structures exhibit, on the average, less molecular complexity (less MW, less number of rings and rotatable bonds), are less hydrophobic (lower CLogP and LogD74), and less druglike (lower druglike scores). These findings indicate that the process of optimizing a lead into a drug results in more complex structures”.
Thus since lead optimization invariably leads to an increase in molecular complexity, molecular weight, and lipophilicity it is highly desirable to weight the screening collection towards smaller, less lipophilic compounds.
Toxicophores
Idiosyncratic drug reactions (IDRs) are a major concern for drug development, they often are not apparent in preclinical toxicity testing and are observed only when the drug is given to large patient populations. The postulated mechanism involves metabolic activation leading a reactive metabolite which can lead to thiol depletion (reaction with glutathione), reversible protein modification (glutathionylation and nitration), further irreversible protein adduct formation and subsequent irreversible protein damage leading to an immune mediated adverse event (Drug Discovery Today Vol. 8, No. 22 November 2003). Similar metabolic activation has been proposed to be responsible for the genotoxicty of some compounds and a variety of toxicophores proposed (Journal of the Iranian Chemical Society, Vol. 2, No. 4, December 2005, pp. 244-267).
Interference with the detection technology
In addition the new screening technologies have introduced new requirements, many now use fluorescence-based technologies and there is a need to avoid compounds that might interfere with the detection technology, or compounds that might chelate Calcium ions.
A number of major pharmaceutical companies have devoted significant resources to evaluating their sample collection in order to identify a “screening collection”. Whilst the precise filters used by each company are confidential Oprea et al (J. Comp. Aid. Mol. Des. 14 (2000) 251-264) have described a “Leadlike” filter.
Suggested criterion for inclusion in the screening collection.
For inclusion in the Screening Collection a molecule can have at most one violation of the following conditions:
1. The number N or O that are hydrogen bond donors must be 5 or less
2. The number of N and O atoms must be 8 or less
3. The molecular weight must be 450 or less
4. The logP must be in the range [-3.5 to 4.5], inclusive
5. The number of rings of size three through eight must be 4 or less
6. The number of rotatable bonds must be 10 or less
In addition the molecule should not contain any of the following reactive groups/toxicophores:-
The molecule must not contain groups in a conservative list of reactive groups: acrylamide, acyclic diketyl, acyl halide/cyanides, activated esters, cyclohexadienes, aldehyde, aliphatic imine, aliphatic ketone, aryl or aliphatic nitro(so)/hydroxylamine, aliphatic (thio)ester, alkyl halide, anhydride, azide, aziridine, beta-heterosubstituted carbonyl, epoxide, halopyrimidine, hetero-allyl, iso(thio)cyanate, maleimide, Michael acceptor, perhalo ketone, phosphonate ester, phospho-, sulfonate ester, thiol, disulphides, thio(urea), O-O single bonds and transition metals.
A structure search would need to be performed to ensure each sample is unique and, depending on the number of samples, a diversity analysis may need to be performed to avoid the collection being biased towards a particular chemotype.
The samples should have a single major component (>95%), and sufficient sample should be immediately available for follow up.
Focused Screening Libraries
The screening collection design described above is aimed at providing a diverse sample collection that can be used to screen against a variety of biological targets. However for some biological targets the nature of the molecular target is well defined and a case can be made for only screening a preselected sub-set of compounds, or even compounds not contained in the screening collection.
Examples might include:-
- Kinase targets
- Well known structural motifs
- Protease Inhibitors
- Cysteine Protease Inhibitors
- Metalloprotease Inhibitors
- Inhaled drugs
- Physicochemical properties may be very different from oral drugs
- Topical targets
- No need for oral activity
- Natural Products
- The structures may not comply with the “rule of five” but they have been a rich source of leads particularly anti-infectives
Ongoing Activities
It should be recognized that the screening collection will not be a static entity and will need to be continuously updated and annotated.
1. Samples will be depleted with use and a decision will need to be made as to whether they should be replaced.
2. Novel chemotypes may become available, these would need to pass the lead-like filters above and be dissimilar to existing compounds.
3. As screens are run promiscuous compounds will be identified and flagged and possibly be removed.
4. New screening technologies may be compromised by some chemotypes.
5. Compounds followed up as leads may reveal unexpected toxicity, this information should be captured and the sample information annotated.
Quality Control
The quality and purity of the screening collection can have an enormous impact on resources when one considers the time wasted following up impure, degraded or incorrectly assigned hits from screening. There are no hard numbers but I’ve heard of cases where up to 40% of hits failed to provide a useful lead due to the poor quality of the screening sample.
Of course QC of a million compound screening library is a significant drain on resources, some companies adopt a strategy of evaluating a random selection on a regular basis, others the entire collection. In both cases, because the vast majority of compounds will never be hits in an assay, resources are being wasted doing QC analysis on “inactive” compounds.
Perhaps a better strategy is to adopt an “In and Hit” protocol, compounds are rigorously evaluated before they are added to the screening collection and then only reassessed if they are a hit in a screen. Any compounds that fail QC as a hit are then removed from the collection. The degree of degradation is very much dependent on the storage conditions and there have been several publications evaluating different conditions.
Worth reading:-
Stability Through the Ages: The GSK Experience, Journal of Biomolecular Screening, Vol. 14, No. 5, 547-556 (2009)
COMDECOM: predicting the lifetime of screening compounds in DMSO solution , Journal of Biomolecular Screening, Vol. 14, No. 5, 557-65 (2009).
Ideally the initial quality control should asses both purity and structural characterization. Samples would be expected to be a single major peak (>95%) by HPLC using two different columns, they would should also have the correct mass. Most of which can be automated. Compounds should also be analysed by 1H NMR however this probably requires a greater degree of human interaction and it may be only possible to do this for the “Hits”.
If you are building up a collection from commercial suppliers then it is worth reading the Chemical Databases and Suppliers page.
Last Updated 20 March 2010
See also Privileged Strictures
See also Fragment Collection
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