Designed Enzymes Precisely Cut Targeted Proteins in Lab Tests

Good news!

"... The researchers specified a target sequence, designed enzymes to cut those targets, and verified their accuracy in doing so across a range of experimental settings. They tested the new enzymes on purified proteins, sequences drawn from proteins implicated in Alzheimer’s disease and amyotrophic lateral sclerosis, and, in one study, inside living human cells cultured in the lab. ...

The functional properties of proteases also enable therapeutic approaches ... These could include destroying a viral protein or a bacterial toxin outright, clearing a misfolded protein that accumulates in neurodegenerative disease, or snipping a protective cap off a drug so it functions only in the tissue where it is needed. ..."

From the abstract (1):

"Despite advances in de novo enzyme design, success has been largely limited to low-energy-barrier model reactions.

Amide bonds such as those linking amino acids along the peptide backbone are stable for hundreds of years in neutral aqueous solution because of the high energy barrier to hydrolysis.

Here we describe the de novo design of enzymes that use an activated cysteine nucleophile to hydrolyse the polypeptide backbone in a sequence-dependent manner, with a success rate of 13/69 = 19% and rate enhancements over the background reaction (kcat/kuncat) of up to 3 × 107.

The designed proteases have folds that are very different from proteases in nature (TM score < 0.50), and six crystal structures are very close to the design models (Cα root mean squared deviation of <1.2 Å), highlighting the capacity for generalization and the accuracy of the design methodology.

Experimental and computational analyses suggest that the remaining gap in activity to the most active native cysteine proteases arises from imperfections in active-site preorganization and substrate positioning.

The designed proteases efficiently cleave their targets in mammalian cells, opening the door to a wide range of synthetic biology applications."

From the abstract (2):

"De novo design of enzymes that cleave amide bonds represents a major challenge.

Here we use RFdiffusion methods to de novo design proteins with water-activating zinc-based active sites adjacent to a peptide substrate binding pocket.

Of 135 designs tested, 36% catalyse cleavage of their cognate peptide substrates at the targeted scissile bond.

The best design accelerates peptide-bond hydrolysis by more than 108-fold relative to the uncatalysed reaction; four substitutions increase this acceleration to more than 1010-fold by improving active-site preorganization.

We use this approach to design metalloproteases that selectively cleave the human disease-associated proteins TDP-43, amyloid-β and serum amyloid A, with rate enhancements of up to 109-fold.

We further design caged cytokines and receptor antagonists that are selectively activated by the designed proteases, enabling bioorthogonal control of cell state. Thus, de novo design can yield highly active, programmable proteases with applications in therapeutics, biotechnology and bioremediation."

Designed Enzymes Precisely Cut Targeted Proteins in Lab Tests - Human Progress

Molecular ‘scissors’ open path to new therapies "In two studies published in Nature, computationally designed enzymes cut disease-related proteins at precise sites."

AI designs working molecular scissors that recognize and cut selected protein targets

De novo design of cysteine proteases (1, no public access)

De novo design of cysteine proteases (1, preprint, open access)

Computational design of metalloproteases (2, no public access)

Computational design of metalloproteases (2, preprint, open access)

Fig. 1 Computational design and experimental testing approach. (1)

Enzyme engineering opens door to novel therapies for Parkinson’s, cancers and other hard-to-target protein diseases

Good news! This could become a powerful new treatment option! This seems to be very promising.

"Now, a new study ... demonstrates a proof of concept for a new strategy: engineering proteases—enzymes that cut proteins at specific sites—to selectively degrade these elusive targets with high precision in the proteome of human cells. ...

the study shows how to reprogram a protease from botulinum toxin to target α-Synuclein—a protein with unstructured regions used here as a model. The study marks one proof point in a broader approach that could be applied to a wide range of targets across the proteome. 

“This work highlights how we can use the power of laboratory evolution to engineer proteases that offer a new way to treat diseases caused by hard-to-target proteins,” ...

To reprogram this precision for α-Synuclein, the research team modified the enzyme using directed evolution, a laboratory process that involves introducing mutations and selecting variants with improved function over multiple cycles.

The result: Protease 5. The challenge, however, wasn’t just reprogramming the protease to target α-Synuclein—it was ensuring that it attacked only α-Synuclein and nothing else. Past attempts to evolve proteases for therapeutic use have resulted in enzymes that targeted too broad a range of proteins, cleaving multiple unintended molecules and causing toxicity in cells.

“α-Synuclein is an incredibly hard protein to target because it doesn’t have a stable structure,”  ...

Although α-Synuclein plays a central role in Parkinson’s disease and related disorders, it was used in this study as a model protein representing a broader class known as intrinsically disordered proteins (IDPs)—proteins that lack a defined shape and are notoriously difficult to target with drugs. This instability makes such illnesses challenging to treat because traditional therapies typically work by attaching to stable pockets on proteins, like a key fitting inside a lock. However, α-Synuclein has no such binding site, leaving few viable treatment options. “That’s where proteases come in,” ... “Instead of needing a specific binding site, they can be engineered to recognize and cut α-Synuclein directly, preventing it from dangerously accumulating in the brain.” 

Using directed evolution, the team stepwise modified the botulinum protease, selecting variants that showed increasing preference for α-Synuclein. “Directed evolution works like selective breeding—just as farmers breed plants for better crops, scientists guide proteins through many small changes, choosing the best version at each step,” ... “Each round of modifications made the enzyme more specialized,” ... “until it could selectively degrade α-Synuclein while leaving other proteins untouched.”

When tested in human cells, Protease 5 nearly eliminated all α-Synuclein proteins, suggesting it could help prevent the harmful buildup seen in Parkinson’s disease. And because the enzyme was designed to precisely target α-Synuclein, it didn’t cause toxicity or disrupt essential cellular functions.  ..."

From the significance and abstract:

"Significance

The ability to evolve proteases that selectively cleave a desired protein in vivo could provide access to useful therapeutic agents.

This would be especially powerful when targeting intrinsically disordered proteins (IDPs), a hard-to-drug class of proteins involved in many human diseases including cancer and neurodegenerative diseases.

Here, we demonstrate the stepwise evolution of clinically used botulinum protease to proteolyze the IDP α-Synuclein which forms plaques in the brains of patients suffering from Parkinson’s disease.

Abstract

There is considerable interest in the targeted degradation of proteins implicated in human disease.

The use of sequence-specific proteases for this purpose is severely limited by the difficulty in engineering the numerous enzyme–substrate interactions required to yield highly selective proteases while maintaining catalytic activity.

Herein, we report a strategy to evolve a protease for the programmed degradation of α-Synuclein, a presynaptic protein closely linked to Parkinson’s disease. Our structure-guided evolution campaign uses the protease from botulinum neurotoxin and showcases the stepwise change of specificity from its native substrate SNAP25 to the selective degradation of α-Synuclein.

The protease’s selectivity is further demonstrated in human cells where near complete degradation of overexpressed human α-Synuclein is observed with no significant effects on cell proliferation. This stepwise strategy may serve as a general approach to evolve highly selective proteases targeting dysregulated proteins."

Enzyme engineering opens door to novel therapies for Parkinson’s, cancers and other hard-to-target protein diseases | Scripps Research "Researchers reprogrammed a botulinum toxin protease to selectively break down disease-causing proteins in human cells, providing proof of concept for developing new therapies for a wide range of illnesses."

Revolutionary Enzyme Engineering Paves the Way for Innovative Treatments in Parkinson’s Disease, Cancer, and Challenging Protein Disorders

Targeted degradation of α-Synuclein using an evolved botulinum toxin protease (no public access)