Synthesis and Optimization of Small Molecule Inhibitors of Prostate Specific Antigen.

Semen liquefaction is a postejaculation process that transforms semen from a gel-like (coagulated) form to a water-like consistency (liquefied). This process is primarily regulated by serine proteases from the prostate gland, most prominently, prostate-specific antigen (PSA; KLK3). Inhibiting PSA activity has the potential to impede liquefaction of human semen, presenting a promising target for nonhormonal contraception in the female reproductive tract. This study employed triazole B1 as a starting compound. Through systematic design, synthesis, and optimization, we identified compound 20 (CDD-3290) as a 216 nM inhibitor of PSA with better stability in media than triazole B1. Further, we also evaluated the selectivity profile of compound 20 (CDD-3290) by testing against closely related proteases and demonstrated excellent inhibition of PSA versus α-chymotrypsin and elastase and similar potency versus thrombin. Thus, compound 20 is an improved PSA inhibitor that can be tested for efficacy in vitro or in the female reproductive tract.

Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro/3CLpro in Living Cells.

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.

Solution-Phase Fmoc-Based Peptide Synthesis for DNA-Encoded Chemical Libraries: Reaction Conditions, Protecting Group Strategies, and Pitfalls.

Peptide drug discovery has shown a resurgence since 2000, bringing 28 non-insulin therapeutics to the market compared to 56 since its first peptide drug, insulin, in 1923. While the main method of discovery has been biological display-phage, mRNA, and ribosome-the synthetic limitations of biological systems has restricted the depth of exploration of peptide chemical space. In contrast, DNA-encoded chemistry offers the synergy of large numbers and ribosome-independent synthetic flexibility for the fast and deeper exploration of the same space. Hence, as a bridge to building DNA-encoded chemical libraries (DECLs) of peptides, we have developed substrate-tolerant amide coupling reaction conditions for amino acid monomers, performed a coupling screen to illustrate such tolerance, developed protecting group strategies for relevant amino acids and reported the limitations thereof, developed a strategy for the coupling of α,α-disubstituted alkenyl amino acids relevant to all-hydrocarbon stapled peptide drug discovery, developed reaction conditions for the coupling of tripeptides likely to be used in DECL builds, and synthesized a fully deprotected DNA-decamer conjugate to illustrate the potency of the developed methodology for on-DNA peptide synthesis.

Geldanamycin-inspired compounds induce direct trans-differentiation of human mesenchymal stem cells to neurons.

Inspired from geldanamycin, the synthesis of a new series of 20-membered macrocyclic compounds is developed. The key features in our design are (i) retention of the fragment having the precise chiral functional groups of geldanamycin at C10, C11, C12 and C14, and (ii) replacement of an olefin moiety with the ester group, and the quinoid sub-structure with the triazole ring. The southern fragment needed for the macrocyclic ring formation was obtained from Evans' syn aldol as the key reaction and with the use of D-mannitol as the cheap source of a chiral starting material. For the synthesis of the northern fragment, we utilized l-ascorbic acid, which provided the desired chiral functional groups at C6 and C7. Further, the chain extension completed the synthesis of the northern fragment. In our approach, the crucial 20 membered macrocyclic ring was formed employing the click chemistry. When tested for their ability to directly trans-differentiate human mesenchymal stem cells to neurons, two novel compounds (20a and 7) from this series were identified and this was further validated by the presence of specific neuronal biomarkers (i.e. nestin, agrin and RTN4).