High-throughput kinetic system for small-molecule drug discovery

Abstract
Evidence is accumulating for higher efficacy of small molecule drugs that have off-rates slower than the
pharmacokinetic clearance rate. Despite the importance of kinetics, the standard approach is low throughput
Biacore testing in de novo drug discovery/screening. But we offer a new way that outruns commercial plate
readers. Capienda Biotech is developing the first instrument offering rapid kinetics for competitive inhibitors at
high throughput for drug discovery and optimization. Advancing upon our NSF SBIR Phase I success in
building a prototype instrument for highly parallel assays and sensitive detection for binding kinase targets (128
tests simultaneously), we propose to upgrade the instrument (up to 384 tests simultaneously) with hardware
upgrade for higher-throughput kinetics. The innovative instrument design and assay chemistry enables us to
characterize compounds that target human Cyclin-Dependent Kinases for oncology. The approach is expected
to apply broadly across a growing portfolio of >500 kinase targets.
Kinase pathways have complex roles in cells. Therapeutic kinase inhibitors are approved to treat cancer or
to modulate the immune system. As of November 2023, the FDA has approved 80 kinase inhibitors: 64 (80%)
for cancer [31]. Twenty cyclin-dependent kinases comprise an important family of targets for oncology, and
CDK subfamilies are committed to either progression through the cell cycle or transcriptional regulation [100].
Unfortunately, despite their potential as oncology targets, CDKs are under-exploited. Only four CDK-specific
drugs have gained FDA approval (abemaciclib, palbociclib, ribociclib, trilaciclib) and all against the same target
subfamily: CDK4/6 [31]. This proposal will expand the members of CDK subfamilies that can be targeted using
high throughput kinetics. The approach will lead Medicinal Chemists to find drugs with greater efficacy and
fewer side-effects due to poor target selectivity.
Selectivity of kinase inhibitors is a major issue in addressing the kinase class of targets. Drugs for auto-
immune and anti-inflammatory indications have been approved by the FDA. Sixteen small molecule inhibitors
target kinases regulating the immune system. Ruxolitinib, baricitinib, momelotinib, tofacitinib and fostamatinib
specifically bind the intended kinases in immune signaling (JAKs or SYK) and have little unintended binding to
typical kinases targeted by oncology drugs [42], all without hematologic abnormalities (neutropenia,
thrombocytopenia or anemia) [51-53,55-56]. By contrast, cancer drugs ponatinib, dasatinib and danusertib
bind their intended targets (BRC-ABL and aurora kinases) and also multiple off-target kinases that regulate the
immune system including BTK, LYN, LCK, FYN and MAPK p38α [42], but in the clinic patients experience
adverse events often including neutropenia, thrombocytopenia, anemia and hypertension [44,46,48].
In the longer run, kinome-wide examination of compounds early on and throughout the drug discovery
process may minimize polypharmacology and the unintended adverse effects in the clinic. These issues would
be ameliorated using a solution for high-throughput selectivity including kinetic characterization.
We will measure several key kinetic parameters: kon, koff, drug residence time, dissociation half-life and
determine Kd. The information we gain will distinguish between promising vs. unpromising compounds.
Measurements like these are needed to find drugs like Gleevec (imatinib), which bind kinase followed by
conformational change of the kinase to lock the inhibitor into a stable inhibitor-kinase complex for long duration
of action and improved pharmacokinetics.
Overall, this project addresses an unmet analytical need in drug discovery, which will open a bottleneck in
the path to new, highly specific medicines. The project will enable kinetic understanding across a large number
of compounds that has been difficult or impractical to attain. We will establish a process for optimization,
validation, workflow and data processing that will distinguish between promising and unpromising compounds.
Our findings are expected to demonstrate a systematic approach to kinetic targeting of kinases. In terms of
kinase selectivity, the approach will also help Medicinal Chemists distinguish between off-targets that are
unimportant (unstable binding) vs. important (stably bound off-target binding by compounds). We will develop
assay chemistry and instruments to facilitate the kinetic approach to drug discovery and lead optimization for
use at pharmaceutical companies. The solution offers a systematic high-throughput approach to discovery and
optimization that will lead to better drugs entering clinical trials.

Grant Number: 1R43TR005404-01
NIH Institute/Center: NIH
Principal Investigator: Mark Bernard