Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins.

Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (M) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target M's catalytic cysteine.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2, the COVID-19 pathogen, relies on its main protease (M) for replication and pathogenesis. M is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as M inhibitors.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome.

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease.

Main protease (M ) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an M substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M catalytic cysteine could be easily synthesized.

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (M ) for replication and pathogenesis. M is a demonstrated target for the development of antivirals for SARS-CoV-2.

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals.

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M inhibitors including PF-07321332 and characterized their M inhibition potency in test tubes (in vitro) and 293T cells (in cellulo).

Repurposing Halicin as a potent covalent inhibitor for the SARS-CoV-2 main protease.

The rapid spread of COVID-19 has caused a worldwide public health crisis. For prompt and effective development of antivirals for SARS-CoV-2, the pathogen of COVID-19, drug repurposing has been broadly conducted by targeting the main protease (M), a key enzyme responsible for the replication of virus inside the host. In this study, we evaluate the inhibition potency of a nitrothiazole-containing drug, halicin, and reveal its reaction and interaction mechanism with M.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (M) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl M inhibitors that contained also an aldehyde warhead and β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with M, their inhibition of M transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors.

A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds.

As one of the most valuable tools for genetic code expansion, pyrrolysyl-tRNA synthetase (PylRS) is structurally related to phenylalanyl-tRNA synthetase (PheRS). By introducing mutations that mimic ligand interactions in PheRS into PylRS, we designed a PylRS mutant. This mutant, designated as oClFRS, recognizes a number of o-substituted phenylalanines for their genetic incorporation at amber codon.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (M) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M inhibition information to assess an M inhibitor.

The -Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors.

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M ) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 -butyl-glycine, and a P4 -terminal -butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M inhibitors including PF-07321332 and characterized their M inhibition potency in test tubes ( ) and human host cells ( ).

The P3 - -Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors.

As an essential enzyme to SARS-CoV-2, main protease (M ) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the -terminal protection group, we synthesized a series of M inhibitors that contain -(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC values that range from 4.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV).

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M ), we have designed and synthesized a series of SC2M inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M active-site cysteine C145.

A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M ), we have designed and synthesized a series of SC2M inhibitors that contain β-( -2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M active site cysteine C145.