Endosomolytic Peptides Enable the Cellular Delivery of Peptide Nucleic Acids.

Precision genetic medicine enlists antisense oligonucleotides (ASOs) to bind to nucleic acid targets important for human disease. Peptide nucleic acids (PNAs) have many desirable attributes as ASOs but lack cellular permeability. Here, we use an assay based on the corrective splicing of an mRNA to assess the ability of synthetic peptides to deliver a functional PNA into a human cell. We find that the endosomolytic peptides L17E and L17ER 4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER 4 enables robust corrective splicing in nearly all treated cells. Peptide-PNA conjugates are even more effective. These results enhance the utility of PNAs as research tools and potential therapeutic agents.

Sitetack: A Deep Learning Model that Improves PTM Prediction by Using Known PTMs.

Post-translational modifications (PTMs) increase the diversity of the proteome and are vital to organismal life and therapeutic strategies. Deep learning has been used to predict PTM locations. Still, limitations in datasets and their analyses compromise success. Here we evaluate the use of known PTM sites in prediction via sequence-based deep learning algorithms. Specifically, PTM locations were encoded as a separate amino acid before sequences were encoded via word embedding and passed into a convolutional neural network that predicts the probability of a modification at a given site. Without labeling known PTMs, our model is on par with others. With labeling, however, we improved significantly upon extant models. Moreover, knowing PTM locations can increase the predictability of a different PTM. Our findings highlight the importance of PTMs for the installation of additional PTMs. We anticipate that including known PTM locations will enhance the performance of other proteomic machine learning algorithms.

Facile Access to Branched Multispecific Proteins.

Approaches that leverage orthogonal chemical reactions to generate protein-protein conjugates have expanded access to bespoke chimeras. Although the literature is replete with examples of the semisynthesis of bispecific proteins, few methods exist for the semisynthesis of protein conjugates of higher complexity (i.e., greater than two-protein fusions). The recent emergence of trispecific cell engagers for immune cell redirection therapies necessitates the development of chemical methods for the construction of trispecific proteins that would otherwise be inaccessible via natural protein synthesis. Here, we demonstrate that 3-bromo-5-methylene pyrrolone (3Br-5MP) can be used to effect the facile chemical synthesis of trispecific peptides and proteins with exquisite control over the addition of each monomer. The multimeric complexes maintain epitope functionality both in human cells and upon immobilization. We anticipate that facile access to trispecific proteins using this 3Br-5MP will have broad utility in basic science research and will quicken the pace of research to establish novel, multimeric immune cell redirection therapies.

Identifying and targeting key driver genes for collagen production within the 11q13/14 breast cancer amplicon.

Genetic studies indicate that breast cancer can be divided into several basic molecular groups. One of these groups, termed IntClust-2, is characterized by amplification of a small portion of chromosome 11 and has a median survival of only five years. Several cancer-relevant genes occupy this portion of chromosome 11, and it is thought that overexpression of a combination of driver genes in this region is responsible for the poor outcome of women in this group. In this study we used a gene editing method to knock out, one by one, each of 198 genes that are located within the amplified region of chromosome 11 and determined how much each of these genes contributed to the survival of breast cancer cells. In addition to well-known drivers such as CCND1 and PAK1 , we identified two different genes ( SERPINH1 and P4HA3 ), that encode proteins involved in collagen synthesis and organization. Using both in vitro and in vivo functional analyses, we determined that P4HA3 and/or SERPINH1 provide a critical driver function on IntClust-2 basic processes, such as viability, proliferation, and migration. Inhibiting these enzymes via genetic or pharmacologic means reduced collagen synthesis and impeded oncogenic signaling transduction in cell culture models, and a small-molecule inhibitor of P4HA3 was effective in treating 11q13 tumor growth in an animal model. As collagen has a well-known association with tissue stiffness and aggressive forms of breast cancer, we believe that the two genes we identified provide an opportunity for a new therapeutic strategy in IntClust-2 breast cancers.

Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye.

The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.

Sensitive detection of SARS-CoV-2 main protease 3CLpro with an engineered ribonuclease zymogen.

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CLpro . Zymogen activation by 3CLpro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications.

Click Organocatalysis: Acceleration of Azide-Alkyne Cycloadditions with Mutually Orthogonal Click Reactions.

"Click organocatalysis" uses mutually orthogonal click reactions to organocatalyze a click reaction. We report the development of an isobenzofuran organocatalyst that increases the rate and regioselectivity of an azide-alkyne cycloaddition. The organocatalytic cycle consists of (1) a Diels-Alder reaction of an alkyne with a diarylisobenzofuran to form a benzooxanorbornadiene, (2) a 1,3-dipolar cycloaddition with an azide to form a 4,5-dihydro-1,2,3-triazole, and (3) a retro-Diels-Alder reaction that releases the triazole product and regenerates the diarylisobenzofuran organocatalyst. The diarylisobenzofuran organocatalyst was computationally designed to catalyze the reaction of perfluorophenyl azide and methyl propiolate to selectively form a 1,4-triazole product. Experimental validation of the designed organocatalyst was obtained with methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl propiolate.

Synthetic Collagen Hydrogels through Symmetric Self-Assembly of Small Peptides.

Animal-sourced hydrogels, such as collagen, are widely used as extracellular-matrix (ECM) mimics in tissue engineering but are plagued with problems of reproducibility, immunogenicity, and contamination. Synthetic, chemically defined hydrogels can avoid such issues. Despite the abundance of collagen in the ECM, synthetic collagen hydrogels are extremely rare due to design challenges brought on by the triple-helical structure of collagen. Sticky-ended symmetric self-assembly (SESSA) overcomes these challenges by maximizing interactions between the strands of the triple helix, allowing the assembly of collagen-mimetic peptides (CMPs) into robust synthetic collagen nanofibers. This optimization, however, also minimizes interfiber contacts. In this work, symmetric association states for the SESSA of short CMPs to probe their increased propensity for interfiber association are modelled. It is found that 33-residue CMPs not only self-assemble through sticky ends, but also form hydrogels. These self-assemblies behave with remarkable consistency across multiple scales and present a clear link between their triple-helical architecture and the properties of their hydrogels. The results show that SESSA is an effective and robust design methodology that enables the rational design of synthetic collagen hydrogels.

Detection of Pulmonary Fibrosis with a Collagen-Mimetic Peptide.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology that is characterized by excessive deposition and abnormal remodeling of collagen. IPF has a mean survival time of only 2-5 years from diagnosis, creating a need to detect IPF at an earlier stage when treatments might be more effective. We sought to develop a minimally invasive probe that could detect molecular changes in IPF-associated collagen. Here, we describe the design, synthesis, and performance of [68Ga]Ga·DOTA-CMP, which comprises a positron-emitting radioisotope linked to a collagen-mimetic peptide (CMP). This peptide mimics the natural structure of collagen and detects irregular collagen matrices by annealing to damaged collagen triple helices. We assessed the ability of the peptide to detect aberrant lung collagen selectively in a bleomycin-induced mouse model of pulmonary fibrosis using positron emission tomography (PET). [68Ga]Ga·DOTA-CMP PET demonstrated higher and selective uptake in a fibrotic mouse lung compared to controls, minimal background signal in adjacent organs, and rapid clearance via the renal system. These studies suggest that [68Ga]Ga·DOTA-CMP identifies fibrotic lungs and could be useful in the early diagnosis of IPF.

Computational study of an oxetane 4H-pyrazole as a Diels-Alder diene.

We combine the effects of spirocyclization and hyperconjugation to increase the Diels-Alder reactivity of the 4H-pyrazole scaffold. A density functional theory (DFT) investigation predicts that 4H-pyrazoles containing an oxetane functionality at the saturated center are extremely reactive despite having a relatively high-lying lowest unoccupied molecular orbital (LUMO) energy.

Differential membrane binding of α/ß-peptide foldamers: implications for cellular delivery and mitochondrial targeting.

The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/ß-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/ß-peptides contained cyclic ß-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides.

Bayesian Inference Elucidates the Catalytic Competency of the SARS-CoV-2 Main Protease 3CLpro.

The main protease of SARS-CoV-2, 3CLpro, is a dimeric enzyme that is indispensable to viral replication and presents an attractive opportunity for therapeutic intervention. Previous reports regarding the key properties of 3CLpro and its highly similar SARS-CoV homologue conflict dramatically. Values of the dimeric Kd and enzymic kcat/KM differ by 106- and 103-fold, respectively. Establishing a confident benchmark of the intrinsic capabilities of this enzyme is essential for combating the current pandemic as well as potential future outbreaks. Here, we use enzymatic methods to characterize the dimerization and catalytic efficiency of the authentic protease from SARS-CoV-2. Specifically, we use the rigor of Bayesian inference in a Markov Chain Monte Carlo analysis of progress curves to circumvent the limitations of traditional Michaelis-Menten initial rate analysis. We report that SARS-CoV-2 3CLpro forms a dimer at pH 7.5 that has Kd = 16 ± 4 nM and is capable of catalysis with kcat = 9.9 ± 1.5 s-1, KM = 0.23 ± 0.01 mM, and kcat/KM = (4.3 ± 0.7) × 104 M-1 s-1. We also find that enzymatic activity decreases substantially in solutions of high ionic strength, largely as a consequence of impaired dimerization. We conclude that 3CLpro is a more capable catalyst than appreciated previously, which has important implications for the design of antiviral therapeutic agents that target 3CLpro.

Fluorescent Guanidinium-Azacarbazole for Oxoanion Binding in Water.

Oxoanions such as carboxylates, phosphates, and sulfates play important roles in both chemistry and biology and are abundant on the cell surface. We report on the synthesis and properties of a rationally designed guanidinium-containing oxoanion binder, 1-guanidino-8-amino-2,7-diazacarbazole (GADAC). GADAC binds to a carboxylate, phosphate, and sulfate in pure water with affinities of 3.6 × 104, 1.1 × 103, and 4.2 × 103 M-1, respectively. Like 2-azacarbazole, which is a natural product that enables scorpions to fluoresce, GADAC is fluorescent in water (λabs = 356 nm, λem = 403 nm, ε = 13,400 M-1 cm-1). The quantum yield of GADAC is pH-sensitive, increasing from Φ = 0.12 at pH 7.4 to Φ = 0.53 at pH 4.0 as a result of the protonation of the aminopyridine moiety. The uptake of GADAC into live human melanoma cells is detectable in the DAPI channel at low micromolar concentrations. Its properties make GADAC a promising candidate for applications in oxoanion binding and fluorescence labeling in biological (e.g., the delivery of cargo into cells) and other contexts.

Bioorthogonal 4H-pyrazole "click" reagents.

4H-Pyrazoles are emerging as useful click reagents. Fluorinating the saturated center enables 4H-pyrazoles to react rapidly as Diels-Alder dienes without a catalyst but compromises the stability of these dienes under physiological conditions. To identify more stable 4H-pyrazoles for bioorthogonal chemistry applications, we investigated the Diels-Alder reactivity and biological stability of three 4-oxo-substituted 4H-pyrazoles. We found that these dienes undergo rapid Diels-Alder reactions with endo-bicyclo[6.1.0]non-4-yne (BCN) while being much more stable to biological nucleophiles than their fluorinated counterparts. We attribute the rapid Diels-Alder reactivity of the optimal oxygen-substituted pyrazole to a combination of antiaromaticity, predistortion, and spirocyclization. Their reactivity and stability suggest that 4-oxo-4H-pyrazoles can be useful bioorthogonal reagents.

Spirocyclization enhances the Diels-Alder reactivities of geminally substituted cyclopentadienes and 4H-pyrazoles.

The Diels-Alder reactivity of 5-membered dienes is tunable through spirocyclization at the saturated center. As the size of the spirocycle decreases, the Diels-Alder reactivity increases with the cyclobutane spirocycle, spiro[3.4]octa-5,7-diene, being the most reactive. Density functional theory calculations suggest that spiro[3.4]octa-5,7-diene dimerizes 220,000-fold faster than 5,5-dimethylcyclopentadiene and undergoes a Diels-Alder reaction with ethylene 1,200-fold faster than 5,5-dimethylcyclopentadiene. These findings show that spirocyclization is an effective way to enhance the Diels-Alder reactivity of geminally substituted 5-membered dienes.

Chemoselective Caging of Carboxyl Groups for On-Demand Protein Activation with Small Molecules.

Tools for on-demand protein activation enable impactful gain-of-function studies in biological settings. Thus far, however, proteins have been chemically caged at primarily Lys, Tyr, and Sec, typically through the genetic encoding of unnatural amino acids. Herein, we report that the preferential reactivity of diazo compounds with protonated acids can be used to expand this toolbox to solvent-accessible carboxyl groups with an elevated pKa value. As a model protein, we employed lysozyme (Lyz), which has an active-site Glu35 residue with a pKa value of 6.2. A diazo compound with a bioorthogonal self-immolative handle esterified Glu35 selectively, inactivating Lyz. The hydrolytic activity of the caged Lyz on bacterial cell walls was restored with two small-molecule triggers. The decaging was more efficient by small molecules than by esterases. This simple chemical strategy was also applied to a hemeprotein and an aspartyl protease, setting the stage for broad applicability.

Modular Diazo Compound for the Bioreversible Late-Stage Modification of Proteins.

We introduce a versatile strategy for the bioreversible modification of proteins. Our strategy is based on a tricomponent molecule, synthesized in three steps, that incorporates a diazo moiety for chemoselective esterification of carboxyl groups, a pyridyl disulfide group for late-stage functionalization with thiolated ligands, and a self-immolative carbonate group for esterase-mediated cleavage. Using cytochrome c (Cyt c) and the green fluorescent protein (GFP) as models, we generated protein conjugates modified with diverse domains for cellular delivery that include a small molecule, targeting and cell-penetrating peptides (CPPs), and a large polysaccharide. As a proof of concept, we used our strategy to effect the delivery of proteins into the cytosol of live mammalian cells in the presence of serum. The cellular delivery of functional Cyt c, which induces apoptosis, highlighted the advantage of bioreversible conjugation on a carboxyl group versus irreversible conjugation on an amino group. The ease and utility of this traceless modification provide new opportunities for chemical biologists.

Inhibition of HIV-1 Protease by a Boronic Acid with High Oxidative Stability.

HIV-1 protease is an important target for pharmaceutical intervention in HIV infection. Extensive structure-based drug design led to darunavir becoming a key chemotherapeutic agent. We replaced the aniline group of darunavir with a benzoxaborolone to form BOL-darunavir. This analogue has the same potency as darunavir as an inhibitor of catalysis by wild-type HIV-1 protease and, unlike darunavir, does not lose potency as an inhibitor of the common D30N variant. Moreover, BOL-darunavir is much more stable to oxidation than is a simple phenylboronic acid analogue of darunavir. X-ray crystallography revealed an extensive network of hydrogen bonds between the enzyme and benzoxaborolone moiety, including a novel direct hydrogen bond from a main-chain nitrogen to the carbonyl oxygen of the benzoxaborolone moiety that displaces a water molecule. These data highlight the utility of benzoxaborolone as a pharmacophore.

Nα -Methylation of arginine: Implications for cell-penetrating peptides.

The field of cell-penetrating peptides is dominated by the use of oligomers of arginine residues. Octanol-water partitioning in the presence of an anionic lipid is a validated proxy for cell-penetrative efficacy. Here, we add one, two, or three N-methyl groups to Ac-Arg-NH2 and examine the effects on octanol-water partitioning. In the absence of an anionic lipid, none of these arginine derivatives can be detected in the octanol layer. In the presence of sodium dodecanoate, however, increasing N-methylation correlates with increasing partitioning into octanol, which is predictive of higher cell-penetrative ability. We then evaluated fully Nα -methylated oligoarginine peptides and observed an increase in their cellular penetration compared with canonical oligoarginine peptides in some contexts. These findings indicate that a simple modification, Nα -methylation, can enhance the performance of cell-penetrating peptides.

Emergent Organoboron Acid Catalysts.

Organoboron acids are stable, organic-soluble Lewis acids with potential application as catalysts for a wide variety of chemical reactions. In this review, we summarize the utility of boronic and borinic acids, as well as boric acid, as catalysts for organic transformations. Typically, the catalytic processes exploit the Lewis acidity of trivalent boron, enabling the reversible formation of a covalent bond with oxygen. Our focus is on recent developments in the catalysis of dehydration, carbonyl condensation, acylation, alkylation, and cycloaddition reactions. We conclude that organoboron acids have a highly favorable prospectus as the source of new catalysts.