Protein kinases are the molecular conductors of our cells. They orchestrate vital signals controlling growth, division, survival, and communication by transferring phosphate groups onto other proteins. But when these conductors malfunction – often stuck in the "on" position – they can drive devastating diseases like cancer, autoimmune disorders, and inflammatory conditions. For decades, drug hunters have focused on blocking the active forms of kinases. Now, a revolutionary strategy is gaining ground: targeting the kinases before they even switch on. This pursuit of "unactivated conformations" promises smarter drugs with fewer side effects.
Why Target the "Off" State?
The Car Analogy
Imagine a kinase like a car. The active state is the engine running in gear, ready to move. Traditional kinase drugs act like jamming a brick under the gas pedal of a car already running – it stops it, but roughly, and might affect other nearby cars (similar kinases) too. This leads to common side effects.
Kinase Conformations
Kinases exist in dynamic shapeshifting states. Crucially, they have distinct "unactivated" or "inactive" conformations. One of the most famous is the "DFG-out" state, named for a trio of amino acids (Asp-Phe-Gly) that flip position, creating a hidden pocket unavailable in the active ("DFG-in") form.
Targeting these unactivated states offers key advantages:
- Sharper Specificity: The hidden pockets in inactive states are often more unique to a particular kinase than the highly conserved active site used by ATP (the kinase's fuel) and traditional inhibitors.
- Overcoming Resistance: Cancer cells often mutate the kinase's active site to evade traditional drugs. Targeting a different, less mutation-prone region in the inactive state can bypass this resistance.
- New Targets: Some disease-causing kinases are notoriously hard to drug conventionally. Their inactive states might present the only viable target.
The Key Experiment: Trapping Src in the Act (of Being Inactive)
A landmark study published in Nature Chemical Biology (2015) by researchers in Kevan Shokat's lab vividly demonstrated the power and potential of targeting unactivated conformations. They focused on Src kinase, a notorious player in several cancers.
Experimental Goal
To definitively prove that a small molecule could selectively bind to and inhibit the DFG-out conformation of Src, and to assess its cellular effects and selectivity profile compared to a traditional active-site inhibitor.
Methodology Step-by-Step
- Designing the "Chemical Probe": The team designed a molecule (later called "compound 1") specifically engineered to fit into the predicted DFG-out pocket of Src.
- Kinase Activity Assays: They tested the ability of compound 1 to inhibit purified Src kinase activity in vitro.
- Selectivity Screening: To check if compound 1 was truly specific, they tested it against a large panel (over 300) of other diverse human kinases.
- Cellular Testing: They treated human cancer cells expressing Src with compound 1 and measured key downstream effects.
- Structural Proof (X-ray Crystallography): They determined the 3D atomic structure of Src bound to compound 1.
Molecular Visualization
Illustration of kinase structure showing active and inactive conformations. The DFG-out state creates a unique binding pocket.
Results and Analysis: A Clear Win for the Inactive State
Key Findings
- Potency & Mechanism Confirmed: Compound 1 potently inhibited Src activity. The X-ray structure irrefutably showed it bound deep within the DFG-out pocket.
- Superior Selectivity: While the traditional active-site inhibitor Bosutinib hit dozens of off-target kinases, compound 1 was exquisitely specific.
- Cellular Efficacy: Compound 1 effectively reduced phosphorylation of Src and its key target FAK in cancer cells.
Cellular Effects
Comparative Analysis
| Feature | Traditional ATP-competitive Inhibitors | Inhibitors of Unactivated Conformations |
|---|---|---|
| Binding Site | ATP pocket (Highly Conserved) | Unique allosteric pockets (e.g., DFG-out pocket) |
| Specificity Potential | Often Low (Many off-target effects) | High (Unique pockets less conserved) |
| Resistance Vulnerability | High (Mutations in ATP pocket common) | Lower (Targets less mutation-prone regions) |
| Target Scope | Many kinases | Includes kinases hard to drug conventionally |
| Example Drug Type | Imatinib (Gleevec), Bosutinib | Gilteritinib (FLT3 DFG-out), Asciminib (ABL1 Myristoyl) |
The Scientist's Toolkit: Hunting Inactive States
Targeting elusive kinase conformations requires specialized tools:
Structure-Based Drug Design Software
Predicts kinase conformations, models drug binding, designs molecules for hidden pockets.
Covalent Probe Libraries
Collections of molecules with reactive "warheads" designed to trap specific inactive states.
Engineered Kinases
Allows creation of mutant kinases sensitive to uniquely designed inhibitors.
Kinase Profiling Panels
Tests drug candidates against hundreds of kinases to assess selectivity.
Cryo-Electron Microscopy
Determines high-resolution structures of large, flexible kinase complexes.
NMR Spectroscopy
Probes kinase dynamics and transient conformational states in solution.
Beyond the Lab: The Future of Kinase Drugs
Current Successes
The success exemplified by the Src study and the subsequent approval of drugs like asciminib (targeting a unique myristoyl pocket in BCR-ABL, another inactive conformation) validates this paradigm shift.
Future Directions
Researchers are now actively hunting for the unique "off" shapes of many other disease-causing kinases. They're developing sophisticated methods to trap these fleeting conformations and design exquisitely selective molecules.
The Goal
Smarter medicines that hit disease targets with pinpoint accuracy, leaving healthy cells untouched, offering patients more effective treatments with dramatically improved quality of life. The hunt for kinases' secret doors is wide open, promising a future of truly targeted therapies.