Robert W. Kirby, Raymond Tang, Louise Webdale and Marc Rogers
Ion channels represent 15 – 20% of historic drug approvals and recent drug discovery projects. Many ion channel families (Nav, Cav, TRPx and GABA) are validated as therapeutic targets based on human genetics, animal models and selective pharmacology. However, ion channels are challenging targets requiring specialist target class knowledge and screening technology such as automated patch clamp (APC) electrophysiology.
Here we outline our example where a pharma company interested in ion channels, but lacking expertise and screening platforms turned to a specialist CRO to fill this knowledge gap. In our case a Japanese pharma company with plate-based assay data wanted to expand medicinal chemistry SAR by accessing high quality APC and ion channel expertise.
During the collaboration selective Kv1.3 modulators with nM potency and efficacy against human T-cells were identified.
Collaboration structure and management
Screening cascade and partner contributions.
The Japanese pharma partner provided the medicinal chemistry SAR which supplied compounds into a 5 tier screening cascade.
Metrion contributed: APC assays using the QPatch system for all tiers. The first tier the primary assessment of potency against hKv1.3 and a gene family member, hKv1.5.
Japanese partner contributed: ADME, PK, ex- and in vivo efficacy and toxicology studies.
Collaboration management
Fast data turn around time
Efficient shipping system and integration into compound management at Metrion ensured rapid data turn around.
Example data turnaround time for the first 30 weekly shipments received from Japan. Metrion adapted its compound handling process to ensure data was returned in a timely manner to keep pace with SAR in Japan. Data for tier 1 assays was returned to partner within 5 working days of compound receipt from Japan.
Consistent pharmacology
Use of positive control allows QC of assay performance and a benchmark to drive SAR.
Consistent pharmacology achieved for positive control used in QPatch Kv1.3 assay. Reproducibility well within industry standard (dashed lines show <3-fold variation) with low week to week variation (red dotted line shows 95% CI). Assay was stable so that potency achieved in week 1 for a specific compound would be repeated when tested 80 weeks later.
Using QPatch to drive robust SAR
Using QPatch to drive SAR meeting potency targets.
Fast data turnaround times coupled with a robust assay assisted meeting medicinal chemistry targets. Shown is a box whisker distribution plot of potency values for compounds grouped per quarter. Initial SAR assessment (Q1) showed good range in potency, however, optimisation of other properties was required (grey Q2 – Q9) before the target potency (IC50 < 0.1 µM) could be achieved (green – Q10 and Q11).
Metrion’s APC expertise used successfully to support the collaboration at each tier
Establishing Kv1.x gene family counter screens (Tier 2)
Important that selectivity of compounds was assessed using the same platform to exclude platform bias. Therefore, full biophysical assessment was performed on QPatch before testing compounds.
Rat Kv1.3 cell line required for cascade (Tier 3)
A rat Kv1.3 cell line was generated as rat models were principally used for in vivo testing (tier 3 and 4 of cascade) and due to the lack of commercial supplier.
Exploring mechanism of action using QPatch (Tier 4)
Placing a variety of cursors on current trace provided important information in translation of data from models:
Extended cardiac panel testing (Tier 5)
After testing for gene family and species selectivity, we assessed any potential cardiac liability by testing compounds in our panel of human cardiac ion channel assays.
Optimised Kv1.3 molecules show nM potency in human T-cells
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The development and validation of electrophysiological assays to study TRPML1 is important to understand the function and pharmacology of the channel. We used a TRPML1 variant that lacks the endo-lysosomal retention sequences (TRPML1-4A), enabling the channel to express at the plasma membrane3. As such channel behaviour can be characterised by means of whole-cell patch-clamp and fluorescence-based techniques.
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