Peptide-Drug Conjugates: An Emerging Direction for the Next Generation of Peptide Therapeutics.

Building on recent advances in peptide science, medicinal chemists have developed a hybrid class of bioconjugates, called peptide-drug conjugates, that demonstrate improved efficacy compared to peptides and small molecules independently. In this Perspective, we discuss how the conjugation of synergistic peptides and small molecules can be used to overcome complex disease states and resistance mechanisms that have eluded contemporary therapies because of their multi-component activity. We highlight how peptide-drug conjugates display a multi-factor therapeutic mechanism similar to that of antibody-drug conjugates but also demonstrate improved therapeutic properties such as less-severe off-target effects and conjugation strategies with greater site-specificity. The many considerations that go into peptide-drug conjugate design and optimization, such as peptide/small-molecule pairing and chemo-selective chemistries, are discussed. We also examine several peptide-drug conjugate series that demonstrate notable activity toward complex disease states such as neurodegenerative disorders and inflammation, as well as viral and bacterial targets with established resistance mechanisms.

Structure-Activity Relationships of the Antimicrobial Peptide Natural Product Apidaecin.

With the growing crisis of antimicrobial resistance, it is critical to continue to seek out new sources of novel antibiotics. This need has led to renewed interest in natural product antimicrobials, specifically antimicrobial peptides. Nonlytic antimicrobial peptides are highly promising due to their unique mechanisms of action. One such peptide is apidaecin (Api), which inhibits translation termination through stabilization of the quaternary complex of the ribosome-apidaecin-tRNA-release factor. Synthetic derivatives of apidaecin have been developed, but structure-guided modifications have yet to be considered. In this work, we have focused on modifying key residues in the Api sequence that are responsible for the interactions that stabilize the quaternary complex. We present one of the first examples of a highly modified Api peptide that maintains its antimicrobial activity and interaction with the translation complex. These findings establish a starting point for further structure-guided optimization of Api peptides.

Mapping the energy landscape of PROTAC-mediated protein-protein interactions.

A principal challenge in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. Recently, growing interest in building predictive models of complexes mediated by Proteolysis Targeting Chimeras (PROTACs) has led to the application of state-of-the-art computational techniques to tackle this problem. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct potential energy and solvation energy landscapes of PROTAC-mediated interactions. Our results suggest that desolvation of amino acids at interfaces could play a dominant role in PROTAC-mediated complex formation.

Synthesis, Optimization, and Structure-Activity Relationships of Imidazo[1,2-a]pyrimidines as Inhibitors of Group 2 Influenza A Viruses.

The influenza A virus (IAV) is a highly contagious virus that causes pandemics and seasonal epidemics, which are major public health issues. Current anti-influenza therapeutics are limited partly due to the continuous emergence of drug-resistant IAV strains; thus, there is an unmet need to develop novel anti-influenza therapies. Here, we present a novel imidazo[1,2-a]pyrimidine scaffold that targets group 2 IAV entry. We have explored three different regions of the lead compound, and we have developed a series of small molecules that have nanomolar activity against oseltamivir-sensitive and -resistant forms of group 2 IAVs. These small molecules target hemagglutinin (HA), which mediates the viral entry process. Mapping a known small-molecule-binding cavity of the HA structure with resistant mutants suggests that these molecules bind to that cavity and block HA-mediated membrane fusion.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

An Innovation 10 Years in the Making: The Stories in the Pages of ACS Medicinal Chemistry Letters.

Innovation in medicinal chemistry has been at the heart of ACS Medicinal Chemistry Letters since the journal's founding 10 years ago. In his inaugural editorial, Editor-in-Chief Dennis Liotta laid out a vision for the journal to become the "premier international journal for rapid communication of cutting-edge studies," and, after 10 years, it has become exactly that. The great hope of drug discovery scientists is that their innovations will lead to new therapeutics to treat unmet medical needs. In the spirit of innovation and in celebration of the recent 10th anniversary of ACS Med. Chem. Lett., we highlight five therapeutics that were first reported or first comprehensively characterized within ACS Med. Chem. Lett.. This overview also serves to introduce the expansion of the scope of the Innovations article type to include Topical Innovations. With this extension, the journal hopes to provide a forum to showcase concise (rather than comprehensive) reviews of topics that are both timely and of great interest to the medicinal chemistry community. Moreover, these articles will emphasize the next steps to move the field toward new areas of interest in medicinal chemistry. Appropriate topics might include case studies of clinical candidates or approved drugs, new assay technologies in drug discovery, novel target classes, and innovative new approaches towards modulation of human physiology. Since its founding 10 years ago, ACS Med. Chem. Lett. has established itself as a venue for the rapid communication of studies in medicinal chemistry and drug discovery. There have been several drugs and clinical candidates that were first reported or first comprehensively characterized in ACS Med. Chem. Lett. In celebration of the 10th anniversary of ACS Med. Chem. Lett. this Topical Innovations article highlights five of these compounds: Ivosidenib, Siponimod, Glasdegib, Parsaclisib, and Dabrafenib.

Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays.

Pharmacologic inhibition of the protein-protein interaction (PPI) interface of the Keap1:Nrf2 complex, which leads to Nrf2 activation and cytoprotective gene expression, offers a promising strategy for disease prevention and treatment. To facilitate identification and validation of small-molecule Keap1:Nrf2 PPI inhibitors in the cellular environment in a low- and medium-throughput manner, we detail two adapted cellular thermal shift assay (CETSA) protocols, Keap1-CETSA, an immunoblotting-based methodology for detecting endogenous Keap1, and Keap1-Glow CETSA, a microtiter plate assay of overexpressed fluorescently-tagged Keap1. For an example of the use and execution of this protocol, please refer to Dayalan Naidu et al. (2021).

The isoquinoline PRL-295 increases the thermostability of Keap1 and disrupts its interaction with Nrf2.

Transcription factor Nrf2 and its negative regulator Keap1 orchestrate a cytoprotective response against oxidative, metabolic, and inflammatory stress. Keap1 is a drug target, with several small molecules in drug development. Here, we show that the isoquinoline PRL-295 increased Keap1 thermostability in lysates from cells expressing fluorescently tagged Keap1. The thermostability of endogenous Keap1 also increased in intact cells and murine liver following PRL-295 treatment. Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET) experiments in cells co-expressing sfGFP-Nrf2 and Keap1-mCherry further showed that PRL-295 prolonged the donor fluorescence lifetime, indicating disruption of the Keap1-Nrf2 protein complex. Orally administered PRL-295 to mice activated the Nrf2transcriptional target NAD(P)H:quinone oxidoreductase 1 (NQO1) in liver and decreased the levels of plasma alanine aminotransferase and aspartate aminotransferase upon acetaminophen-induced hepatic injury. Thus, PRL-295 engages the Keap1 protein target in cells and in vivo, disrupting its interaction with Nrf2, leading to activation of Nrf2-dependent transcription and hepatocellular protection.

A recombinant Cedar virus based high-throughput screening assay for henipavirus antiviral discovery.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic, bat-borne paramyxoviruses in the genus Henipavirus that cause severe and often fatal acute respiratory and/or neurologic diseases in humans and livestock. There are currently no approved antiviral therapeutics or vaccines for use in humans to treat or prevent NiV or HeV infection. To facilitate development of henipavirus antivirals, a high-throughput screening (HTS) platform was developed based on a well-characterized recombinant version of the nonpathogenic Henipavirus, Cedar virus (rCedV). Using reverse genetics, a rCedV encoding firefly luciferase (rCedV-Luc) was rescued and its utility evaluated for high-throughput antiviral compound screening. The luciferase reporter gene signal kinetics of rCedV-Luc in different human cell lines was characterized and validated as an authentic real-time measure of viral growth. The rCedV-Luc platform was optimized as an HTS assay that demonstrated high sensitivity with robust Z' scores, excellent signal-to-background ratios and coefficients of variation. Eight candidate compounds that inhibited rCedV replication were identified for additional validation and demonstrated that 4 compounds inhibited authentic NiV-Bangladesh replication. Further evaluation of 2 of the 4 validated compounds in a 9-point dose response titration demonstrated potent antiviral activity against NiV-Bangladesh and HeV, with minimal cytotoxicity. This rCedV reporter can serve as a surrogate yet authentic BSL-2 henipavirus platform that will dramatically accelerate drug candidate identification in the development of anti-henipavirus therapies.

Molecular basis for the disruption of Keap1-Nrf2 interaction via Hinge & Latch mechanism.

The Keap1-Nrf2 system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. One model for the molecular mechanisms leading to Nrf2 activation is the Hinge-Latch model, where the DLGex-binding motif of Nrf2 dissociates from Keap1 as a latch, while the ETGE motif remains attached to Keap1 as a hinge. To overcome the technical difficulties in examining the binding status of the two motifs during protein-protein interaction (PPI) simultaneously, we utilized NMR spectroscopy titration experiments. Our results revealed that latch dissociation is triggered by low-molecular-weight Keap1-Nrf2 PPI inhibitors and occurs during p62-mediated Nrf2 activation, but not by electrophilic Nrf2 inducers. This study demonstrates that Keap1 utilizes a unique Hinge-Latch mechanism for Nrf2 activation upon challenge by non-electrophilic PPI-inhibiting stimuli, and provides critical insight for the pharmacological development of next-generation Nrf2 activators targeting the Keap1-Nrf2 PPI.

A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ.

The lifelong relationship between the Hawaiian bobtail squid Euprymna scolopes and its microbial symbiont Vibrio fischeri represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. "Biofilm-up" and "biofilm-down" mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in V. fischeri and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors.IMPORTANCE The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

Charting the sequence-activity landscape of peptide inhibitors of translation termination.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

SAR Study on Estrogen Receptor α/ß Activity of (Iso)flavonoids: Importance of Prenylation, C-Ring (Un)Saturation, and Hydroxyl Substituents.

Many botanicals used for women's health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERß) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure-activity relationship study of (iso)flavonoids was conducted to identify ERß-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2-C3 unsaturation resulted in increased ERß potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50 (ERß): 0.0035 ± 0.00040 µM], whereas 4'-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50 (ERß): 1.7 ± 0.70 µM]. However, nonprenylated and C2-C3 unsaturated isoflavonoids showed increased ERß estrogenic activity [e.g., genistein, EC50 (ERß): 0.0022 ± 0.0004 µM]. Licorice (Glycyrrhiza inflata, [EC50 (ERα): 1.1 ± 0.20; (ERß): 0.60 ± 0.20 µg/mL], containing 8-PA, and red clover [EC50 (ERα): 1.8 ± 0.20; (ERß): 0.45 ± 0.10 µg/mL], with genistein, showed ERß-preferential activity as opposed to hops [EC50 (ERα): 0.030 ± 0.010; (ERß): 0.50 ± 0.050 µg/mL] and Epimedium sagittatum [EC50 (ERα): 3.2 ± 0.20; (ERß): 2.5 ± 0.090 µg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERß-preferential flavonoids could plausibly contribute to ERß-protective benefits in menopausal women.

Synthesis and Evaluation of Noncovalent Naphthalene-Based KEAP1-NRF2 Inhibitors.

The oxidative stress response, gated by the protein-protein interaction of KEAP1 and NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may operate through several biological pathways rather than solely through the activation of the oxidative stress response. Recently, our lab has reported a nonelectrophilic, monoacidic, naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a detailed structure-activity relationship of naphthalene-based NRF2 activators, an X-ray crystal structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the NRF2 binding pocket of KEAP1.

Scaffold-hopping as a strategy to address metabolic liabilities of aromatic compounds.

Understanding and minimizing oxidative metabolism of aromatic compounds is a key hurdle in lead optimization. Metabolic processes not only clear compounds from the body, but they can also transform parent compounds into reactive metabolites. One particularly useful strategy when addressing metabolically labile or oxidation-prone structures is scaffold-hopping. Replacement of an aromatic system with a more electron-deficient ring system can often increase robustness towards cytochrome P450-mediated oxidation while conserving the structural requirements of the pharmacophore. The most common example of this substitution strategy, replacement of a phenyl ring with a pyridyl substituent, is prevalent throughout the literature; however scaffold-hopping encompasses a much wider scope of heterocycle replacement. This review will showcase recent examples where different scaffold-hopping approaches were used to reduce metabolic clearance or block the formation of reactive metabolites. Additionally, we will highlight considerations that should be made to garner the most benefit from a scaffold-hopping strategy for lead optimization.

Isoquinoline Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-like 2 (KEAP1-NRF2) Inhibitors with High Metabolic Stability.

Pharmacological activation of NRF2 (nuclear factor erythroid 2-related factor 2) arises from blocking the interaction of NRF2 with its negative regulator, KEAP1 (Kelch-like ECH-associated protein 1). We previously reported an isoquinoline-based NRF2 activator, but this compound showed negative logD7.4 and a -2 charge at physiological pH, which may have limited its membrane permeability. In this work, we report potent, metabolically stable analogs that result from replacing a carboxymethyl group at the 4-position with a fluoroalkyl group.

Steroid receptor/coactivator binding inhibitors: An update.

The purpose of this review is to highlight recent developments in small molecules and peptides that block the binding of coactivators to steroid receptors. These coactivator binding inhibitors bind at the coregulator binding groove, also known as Activation Function-2, rather than at the ligand-binding site of steroid receptors. Steroid receptors that have been targeted with coactivator binding inhibitors include the androgen receptor, estrogen receptor and progesterone receptor. Coactivator binding inhibitors may be useful in some cases of resistance to currently prescribed therapeutics. The scope of the review includes small-molecule and peptide coactivator binding inhibitors for steroid receptors, with a particular focus on recent compounds that have been assayed in cell-based models.