BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 µM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 µM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

Taf2 mediates DNA binding of Taf14.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription.

Accurate Mass Identification of an Interfering Water Adduct and Strategies in Development and Validation of an LC-MS/MS Method for Quantification of MPI8, a Potent SARS-CoV-2 Main Protease Inhibitor, in Rat Plasma in Pharmacokinetic Studies.

MPI8, a peptidyl aldehyde, is a potent antiviral agent against coronavirus. Due to unique tri-peptide bonds and the formyl functional group, the bioassay of MPI8 in plasma was challenged by a strong interference from water MPI8. Using QTOF LC-MS/MS, we identified MPI8•H2O as the major interference form that co-existed with MPI8 in aqueous and biological media. To avoid the resolution of MPI8 and MPI8•H2O observed on reverse phase columns, we found that a Kinetex hydrophilic interaction liquid chromatography (HILIC) column provided co-elution of both MPI8 and MPI8•H2O with a good single chromatographic peak and column retention of MPI8 which is suitable for quantification. Thus, a sensitive, specific, and reproducible LC-MS/MS method for the quantification of MPI8 in rat plasma was developed and validated using a triple QUAD LC-MS/MS. The chromatographic separation was achieved on a Kinetex HILIC column with a flow rate of 0.4 mL/min under gradient elution. The calibration curves were linear (r2 > 0.99) over MPI8 concentrations from 0.5−500 ng/mL. The accuracy and precision are within acceptable guidance levels. The mean matrix effect and recovery were 139% and 73%, respectively. No significant degradation of MPI8 occurred under the experimental conditions. The method was successfully applied to a pharmacokinetic study of MPI8 after administration of MPI8 sulfonate in rats.

Zinc thiotropolone combinations as inhibitors of the SARS-CoV-2 main protease.

Numerous organic molecules are known to inhibit the main protease of SARS-CoV-2, (SC2Mpro), a key component in viral replication of the 2019 novel coronavirus. We explore the hypothesis that zinc ions, long used as a medicinal supplement and known to support immune function, bind to the SC2Mpro enzyme in combination with lipophilic tropolone and thiotropolone ligands, L, block substrate docking, and inhibit function. This study combines synthetic inorganic chemistry, in vitro protease activity assays, and computational modeling. While the ligands themselves have half maximal inhibition concentrations, IC50, for SC2Mpro in the 8-34 µM range, the IC50 values are ca. 100 nM for Zn(NO3)2 which are further enhanced in Zn-L combinations (59-97 nM). Isolation of the Zn(L)2 binary complexes and characterization of their ability to undergo ligand displacement is the basis for computational modeling of the chemical features of the enzyme inhibition. Blind docking onto the SC2Mpro enzyme surface using a modified Autodock4 protocol found preferential binding into the active site pocket. Such Zn-L combinations orient so as to permit dative bonding of Zn(L)+ to basic active site residues.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV.

With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. While little is known about the virus, an examination of the genome sequence shows strong homology with its more well-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations which may hamper efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RdRp and 3CLpro, share a strikingly high (>95%) homology to SARS-CoV. Herein, we suggest 4 potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that can be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad spectrum anti-coronaviral agents for future epidemics.

Stepwise Assembly of Turn-on Fluorescence Sensors in Multicomponent Metal-Organic Frameworks for in†Vitro Cyanide Detection.

The controlled synthesis of multicomponent metal-organic frameworks (MOFs) allows for the precise placement of multiple cooperative functional groups within a framework, leading to emergent synergistic effects. Herein, we demonstrate that turn-on fluorescence sensors can be assembled by combining a fluorophore and a recognition moiety within a complex cavity of a multicomponent MOF. An anthracene-based fluorescent linker and a hemicyanine-containing CN- -responsive linker were sequentially installed into the lattice of PCN-700. The selective binding of CN- to hemicyanine inhibited the energy transfer between the two moieties, resulting in a fluorescence turn-on effect. Taking advantage of the high tunability of the MOF platform, the ratio between anthracene and the hemicyanine moiety could be fine-tuned in order to maximize the sensitivity of the overall framework. The optimized MOF-sensor had a CN- -detection limit of 0.05 µm, which is much lower than traditional CN- fluorescent sensors (about 0.2 µm).

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

Structural insights into the π-π-π stacking mechanism and DNA-binding activity of the YEATS domain.

The YEATS domain has been identified as a reader of histone acylation and more recently emerged as a promising anti-cancer therapeutic target. Here, we detail the structural mechanisms for π-π-π stacking involving the YEATS domains of yeast Taf14 and human AF9 and acylated histone H3 peptides and explore DNA-binding activities of these domains. Taf14-YEATS selects for crotonyllysine, forming π stacking with both the crotonyl amide and the alkene moiety, whereas AF9-YEATS exhibits comparable affinities to saturated and unsaturated acyllysines, engaging them through π stacking with the acyl amide. Importantly, AF9-YEATS is capable of binding to DNA, whereas Taf14-YEATS is not. Using a structure-guided approach, we engineered a mutant of Taf14-YEATS that engages crotonyllysine through the aromatic-aliphatic-aromatic π stacking and shows high selectivity for the crotonyl H3K9 modification. Our findings shed light on the molecular principles underlying recognition of acyllysine marks and reveal a previously unidentified DNA-binding activity of AF9-YEATS.

Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics.

Using Amber and Ochre Nonsense Codons to Code Two Different Noncanonical Amino Acids in One Protein Gene.

In Escherichia coli, conventional amber and ochre stop codons can be separately targeted by engineered amber-suppressing Methanocaldococcus jannaschii tyrosyl-tRNA synthetase-tRNAPyl and ochre-suppressing Methanosarcina maezi pyrrolysyl-tRNA synthetase-tRNAPyl pairs for coding two different noncanonical amino acids in one protein gene. Here, we describe the application of this approach to produce a protein with two distinct chemical functionalites which can be selectively labeled with two fluorescent dyes.

tRNAPyl: Structure, function, and applications.

Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNAPyl). The secondary structure of tRNAPyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNAPyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNAPyl, its interaction with PylRS, and survey recent progress on the use of tRNAPyl as a tool for genetic code expansion.

Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids.

By evolving the N-terminal domain of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNAPyl , a mutant clone displaying improved amber-suppression efficiency for the genetic incorporation of Nϵ -(tert-butoxycarbonyl)-l-lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ -acetyl-l-lysine and Nϵ -(4-azidobenzoxycarbonyl)-l-δ,ϵ-dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N-terminal domain of PylRS that was separated from its catalytic domain for charging tRNAPyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system-based noncanonical amino-acid mutagenesis.

Proteins with Site-Specific Lysine Methylation.

As an important epigenetic mark, lysine methylations play critical roles in the regulation of both chromatin and non-chromatin proteins. There are three levels of lysine methylation, mono-, di-, and trimethylation. Each one has turned out to be biologically distinctive. For the biochemical characterization of proteins with lysine methylation, multiple chemical biology methods have been developed. This concept article will highlight these developments and their applications in epigenetic investigation of protein functions.

Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking.

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein-protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein-protein interactions and their interfaces in living cells.

A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids.

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, ß-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.

A Versatile Approach for Site-Specific Lysine Acylation in Proteins.

Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

A Genetically Encoded Allysine for the Synthesis of Proteins with Site-Specific Lysine Dimethylation.

Using the amber suppression approach, Nϵ -(4-azidobenzoxycarbonyl)-δ,ϵ-dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site-specific lysine dimethylation. Using this approach, dimethyl-histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.