Pentelute Lab MIT | Pentelute Lab MIT - Research Page
The Pentelute Lab leverages expertise in peptide chemistry, molecular biology, and technology development to create peptide and protein-based therapeutics and tools for chemical biology.
Pentelute Lab, MIT, Cambridge, Chemistry, Molecular biology, technology development, peptide, protein-based therapeutics, chemical Biology
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RESEARCH

Throughout evolution, Nature has developed molecular machines to rapidly manufacture, tailor, and deliver large functional biopolymers such as proteins into specific cells. Inspired by these mechanisms of Nature, the Pentelute Lab has aimed to invent new chemistry for the efficient and selective modification of proteins, to ‘hijack’ these biological machines for efficient drug delivery into cells and to create new machines to rapidly and efficiently manufacture peptides and proteins.

 

A main goal of the Pentelute Lab is to invent new chemistry to modify Nature’s proteins to enhance their therapeutic properties for human medicine. This goal has posed immense challenges because proteins contain 20 amino acids that present different reactive functional groups and have a 3D shape that is moderately stable. In light of this, the newly developed chemistry needs to be protein compatible, site-selective, quantitative, and carried out in water at reasonable temperatures to maintain protein integrity and function. The Pentelute Lab has met these challenges and has developed a series of highly efficient and selective chemistries that can modify the amino acid cysteine and lysine within peptides and proteins. These newly developed chemistries can be catalyzed by enzymes or even promoted by a motif discovered by Pentelute’s group, which is coined a ‘pi-clamp’.  This extensive protein modification toolkit has enabled the production of some powerful molecules including peptide macrocycles that cross cell membranes to disrupt cancer or antibody drug conjugates to kill breast cancer cells.

 

The Pentelute group is also focused on the delivery of large biomolecules into the cell cytosol. The group has developed a chemical approach for the systematic investigation of a nontoxic form of anthrax toxin, which transports enzymes into cells via a protective antigen-protein pump.  The Pentelute Lab has recently discovered that the protein pump can deliver a wide range of cargo molecules into cells including antibody mimics, mirror-image proteins, small molecules, and enzymes. Once in the cytosol, the cargo activates biologically and in certain cases perturbs protein-protein interactions that drive cancer. The Pentelute group made a noteworthy cell biology discovery with this biomolecular delivery platform: the act of simply installing a single D-amino acid on an otherwise large L-protein turns off a key mechanism for cytosolic protein degradation. This discovery will aid in the development of durable cell-based protein therapeutics.

 

The Pentelute group, a protein and peptide focused lab, has also invented a fully automated fast-flow machine to accelerate the chemical manufacture of polypeptides. It has built the world’s fastest and most efficient machine that can produce thousands of amide-bonds orders of magnitude faster than commercially available instruments. The machine is inspired by Nature’s ribosome that can incorporate 9 amino acids into a polypeptide chain per second. While the Pentelute group’s fast-flow technology is not as fast as the ribosome, it can form one amide bond in seconds. This technology not only facilitates rapid polypeptide generation but it has enabled the group to carry out an entire D-scan of proteins to investigate protein folding and functions. This technology may solve the manufacturing problem for personalized peptide cancer vaccines.

Selective peptide & protein arylation

Can new arylation chemistry for efficient & selective modification of unprotected peptides & proteins be developed?

In the past five years, the Pentelute lab has discovered a number of approaches for robust arylation of biomolecules. These chemistries have found multiple applications in peptide macrocyclization and synthesis of antibody-drug conjugates.

Perfluoroarylation of unprotected peptides

Will cysteine containing peptides react with various perfluoroarenes to form macrocyclic peptides?

Highly efficient macrocyclization of unprotected peptides was achieved. Novel compounds show enhanced cell penetration, increased stability to proteases, and tunable structural properties.

 

(A. Spokoyny et al. JACS., 2013, 135, 5946-5949)

Enzyme-catalyzed arylation

Can perfluoroarylation reactions be aided by enzymatic catalysis in water?

Glutathione S-transferase efficiently catalyzed the regiospecific arylation at cysteine in peptides and proteins containing a GSH tag. This chemistry was further developed to perform macrocyclization of long, fully unprotected peptides in water.

(C. Zhang et al. Angew. Chem Int. Ed., 2013, 52, 14001–14005;

C. Zhang et al. Org. Lett., 2014, 16, 3652–3655)

Site-selective cysteine arylation

Can a motif which allows for site-selective, specific labeling of a cysteine residue in an antibody protein be discovered?

A π-clamp was discovered that enables the preparation of homogeneous and functional site-specific antibody drug conjugates that killed breast cancer cells. π-clamp has yet to be observed in Nature.

(C. Zhang et al. Nat. Chem., 2016, 8, 120-128)

Salt effect in π-clamp arylation

Can site-selective cysteine arylation be further accelerated by changing the reaction conditions?

Performing reaction in high salt buffers greatly improves the rate of labeling by four orders of magnitude. Antibody-drug conjugates prepared maintained full biological activity. Computational studies were done collaboratively with Prof. Van Voorhis Lab.

(P. Dai et al. ACS Cent. Sci., 2016, 2, 637–646)

Palladium Arylation of Proteins

Can generic arenes be utilized for cysteine bioconjugation?

Palladium complexes proved to be excellent reagents for cysteine modification. Reaction is complete in seconds and shows broad substrate scope. The chemistry was applied to prepare linker-free antibody-drug conjugates. This is a collaborative project with Prof. Buchwald Lab.

(E. Vinogradova et al. Nature, 2015, 526, 687-691)

Lysine Perfluoroarylation

Can the toolbox of arylation chemistry be expanded to other amino acids?

A plethora of novel arylation agents allows facile lysine arylation of unprotected peptides. This chemistry was used to develop a new class of macrocyclic peptides with improved stability and excellent cell penetration.

(G. Lautrette et al. JACS, 2016, 138, 8340-8343)

Anthrax toxin mediated cytosolic delivery of macromolecules

What types of unnatural bioactive molecules can be translocated efficiently?

Over the past five years, Pentelute lab demonstrated that Anthrax toxin system can be leveraged to deliver a variety of natural and unnatural macromolecules. These delivered molecules were functional inside the cytosol.

(A. Rabideau et al. ACS Chem. Bio., 2016, 11, 1490–1501)

Preparation of structurally diverse LFN conjugates

Can a chemistry toolbox for facile modification of LFn be developed?

A number of different chemical techniques enable facile conjugation of chemically diverse biomolecules to LFN; 100’s conjugates have been prepared.

(R. Policarpo et al. ACIE, 2014, 53, 9203-9208; J. Ling et al. JACS, 2012, 134, 10749-10752)

Delivery of antibody mimetics

Can delivery of biologically active antibody mimetics lead to efficient perturbations of key protein-protein interactions?

Functional antibody mimics were successfully delivered and shown to perturb cancer relevant protein-protein interactions. The system substantially outperformed established CPP techniques for delivery by at least four orders of magnitude.

(X. Liao et al. ChemBioChem, 2014, 15, 2458 – 2466)

Delivery of mirror image proteins and peptides

Will PA accommodate translocation of protein enantiomers?
Will these biomolecules show bioactivity in the cytosol?

For the first time, robust delivery into cells of D-proteins was achieved. A D-peptide was delivered into brain cancer cells and shown to perturb a cancer relevant protein-protein interaction.

(A. Rabideau et al. Chem. Sci., 2015, 6, 648 – 653)

Delivery of small molecule therapeutics

Can bulky small molecules with non-peptidic backbones be delivered to the cytosol?

Multiple molecules with non-peptidic backbone or peptides with unnatural side chains were successfully delivered, pushing the limits of PA pore translocation.

(A. Rabideau et al. Sci. Rep., 2015, 5:11944)

Increasing cytosolic stability of LFN-conjugates

Is it possible to enhance the stability of delivered proteins?

For this first time, it was shown that the simple addition of just one D-amino acid to the N-terminus of a protein significantly increased its cytosolic stability by orders of magnitude.

(A. Rabideau et al. ACS Cent. Sci., 2015, 1, 423-430)

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