Simultaneous monitoring of eight human respiratory viruses including SARS-CoV-2 using liquid chromatography-tandem mass spectrometry.

Diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has primarily been achieved using reverse transcriptase polymerase chain reaction (RT-PCR) for acute infection, and serology for prior infection. Assay with RT-PCR provides data on presence or absence of viral RNA, with no information on virus replication competence, infectivity, or virus characterisation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is typically not used in clinical virology, despite its potential to provide supplemental data about the presence of viral proteins and thus the potential for replication-competent, transmissible virus. Using the SARS-CoV-2 as a model virus, we developed a fast 'bottom-up' proteomics workflow for discovery of target virus peptides using 'serum-free' culture conditions, providing high coverage of viral proteins without the need for protein or peptide fractionation techniques. This workflow was then applied to Coronaviruses OC43 and 229E, Influenza A/H1N1 and H3N2, Influenza B, and Respiratory Syncytial Viruses A and B. Finally, we created an LC-MS/MS method for targeted detection of the eight-virus panel in clinical specimens, successfully detecting peptides from the SARS-CoV-2 ORF9B and nucleoprotein in RT-PCR positive samples. The method provides specific detection of respiratory viruses from clinical samples containing moderate viral loads and is an important further step to the use of LC-MS/MS in diagnosis of viral infection.

Cyclic Peptides as Drugs for Intracellular Targets: The Next Frontier in Peptide Therapeutic Development.

Developing macrocyclic peptides that can reach intracellular targets is a significant challenge. This review discusses the most recent strategies used to develop cell permeable cyclic peptides that maintain binding to their biological target inside the cell. Macrocyclic peptides are unique from small molecules because traditional calculated physical properties are unsuccessful for predicting cell membrane permeability. Peptide synthesis and experimental membrane permeability is the only strategy that effectively differentiates between cell permeable and cell impermeable molecules. Discussed are chemical strategies, including backbone N-methylation and stereochemical changes, which have produced molecular scaffolds with improved cell permeability. However, these improvements often come at the expense of biological activity as chemical modifications alter the peptide conformation, frequently impacting the compound's ability to bind to the target. Highlighted is the most promising approach, which involves side-chain alterations that improve cell permeability without impact binding events.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs.

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

Synthesis and Structure-Activity Relationships of Inhibitors That Target the C-Terminal MEEVD on Heat Shock Protein 90.

Herein, we describe the synthesis and structure-activity relationships of cyclic peptides designed to target heat shock protein 90 (Hsp90). Generating 19 compounds and evaluating their binding affinity reveals that increasing electrostatic interactions allows the compounds to bind more effectively with Hsp90 compared to the lead structure. Exchanging specific residues for lysine improves binding affinity for Hsp90, indicating some residues are not critical for interacting with the target, whereas others are essential. Replacing l- for d-amino acids produced compounds with decreased binding affinity compared to the parent structure, confirming the importance of conformation and identifying key residues most important for binding. Thus, a specific conformation and electrostatic interactions are required in order for these inhibitors to bind to Hsp90.

Improving the Cell Permeability of Polar Cyclic Peptides by Replacing Residues with Alkylated Amino Acids, Asparagines, and d-Amino Acids.

The design, synthesis, and cell permeability of 19 hydrophilic macrocyclic peptides is presented. By systematically analyzing the impact of three different approaches (alkylated amino acids, asparagines, and d-amino acids) on the permeability of polar peptides, a well-defined strategy for optimizing cell permeability is provided. These three new methods can be used individually or in combination to effectively convert polar peptides into cell permeable molecules, and the results can be applied to the rapidly expanding peptide therapeutic industry.

Hitting a Moving Target: How Does an N-Methyl Group Impact Biological Activity?

Macrocycles have several advantages over small-molecule drugs when it comes to addressing specific protein-protein interactions as therapeutic targets. Herein we report the synthesis of seven new cyclic peptide molecules and their biological activity. These macrocycles were designed to understand how moving an N-methyl moiety around the peptide backbone impacts biological activity. Because the lead non-methylated structure inhibits the oncogenic regulator heat-shock protein 90 (Hsp90), two of the most potent analogues were evaluated for their Hsp90 inhibitory activity. We show that incorporating an N-methyl moiety controls the conformation of the macrocycle, which dramatically impacts cytotoxicity and binding affinity for Hsp90. Thus, the placement of an N-methylated amino acid within a macrocycle generates an unpredictable change to the compound's conformation and hence biological activity.

Regulating the master regulator: Controlling heat shock factor 1 as a chemotherapy approach.

Described is the role that heat shock factor 1 (HSF1) plays in regulating cellular stress. Focusing on the current state of the HSF1 field in chemotherapeutics we outline the cytoprotective role of HSF1 in the cell. Summarizing the mechanism by which HSF1 regulates the unfolded proteins that are generated under stress conditions provides the background on why HSF1, the master regulator, is such an important protein in cancer cell growth. Summarizing siRNA knockdown results and current inhibitors provides a comprehensive evaluation on HSF1 and its current state. One set of molecules stands out, in that they completely obliterate the levels of HSF1, while simultaneously inhibiting heat shock protein 90 (Hsp90). These molecules are extremely promising as chemotherapeutic agents and as tools that may ultimately provide the connection between Hsp90 inhibition and HSF1 protein levels.

Chemically accessible hsp90 inhibitor that does not induce a heat shock response.

Recent cancer therapies have focused on targeting biology networks through a single regulatory protein. Heat shock protein 90 (hsp90) is an ideal oncogenic target as it regulates over 400 client proteins and cochaperones. However, clinical inhibitors of hsp90 have had limited success; the primary reason being that they induce a heat shock response. We describe the synthesis and biological evaluation of a new hsp90 inhibitor, SM253. The previous generation on which SM253 is based (SM145) has poor overall synthetic yields, low solubility, and micromolar cytotoxicity. By comparison SM253 has relatively high overall yields, good aqueous solubility, and is more cytotoxic than its parent compound. Verification that hsp90 is SM253's target was accomplished using pull-down and protein folding assays. SM253 is superior to both SM145 and the clinical candidate 17-AAG as it decreases proteins related to the heat shock response by 2-fold, versus a 2-4-fold increase observed when cells are treated with 17-AAG.