Department of Pharmaceutical Sciences, UCI

This paper reports highly active analogues of clovibactin in which the rare, noncanonical amino acid d-hydroxyasparagine is replaced with the commercially available amino acid d-threonine. Sequential mutation of leucines 2, 7, and 8 to the more hydrophobic homologue cyclohexylalanine dramatically increases the antibiotic activity of d-Thr₅-clovibactin. The resulting analogues (d-Cha₂,d-Thr₅-clovibactin, Cha₇,d-Thr₅-clovibactin, and Cha₈,d-Thr₅-clovibactin) are readily prepared by standard peptide synthesis techniques and exhibit excellent activity (≤1 μg/mL) against the Gram-positive, drug-resistant pathogens MRSA and VRE.

The architecture of cells and the tissue they form within multicellular organisms are highly complex and dynamic. Cells optimize their function within tissue microenvironments by expressing specific subsets of RNAs. Advances in cell tagging methods enable spatial understanding of RNA expression when merged with transcriptomics. However, these techniques are currently limited by the spatial resolution of the tagging, the number of RNAs that can be sequenced, and multiplexing to isolate spatially-distinct cells within the same tissue landscape. To address these limitations, we developed CrossSeq, which employs photocrosslinking fluorescent probes and confocal microscopy activation to demarcate user-defined regions of interest on fixed cells for multiplexed spatial transcriptomic analysis. We investigate phenyl azide and diazirine crosslinking scaffolds and define their photoactivity profiles. We then deploy the aryl azide scaffold with three fluorophores for multiplexing on glyoxal fixed cells and analyze the defined populations using flow cytometry. Finally, we apply CrossSeq to investigate an in vitro MDA-MB-231-LM2 metastatic cancer migration model to evaluate changes in gene expression at the migratory cell front versus the exterior population. We anticipate this new technology will be a valuable tool addition as it will enable easier access to spatial transcriptomic analysis for the scientific community using conventional microscopy and analysis techniques.

Loss-of-function sequence variants in KCNA1, which encodes the voltage-gated potassium channel Kv1.1, cause Episodic Ataxia Type 1 (EA1) and epilepsy. Due to a paucity of drugs that directly rescue mutant Kv1.1 channel function, current therapeutic strategies for KCNA1-linked disorders involve indirect modulation of neuronal excitability. Native Americans have traditionally used conifer extracts to treat paralysis, weakness, and pain, all of which may involve altered electrical activity and/or Kv1.1 dysfunction specifically. Here, screening conifer extracts, we found that Chamaecyparis pisifera increases wild-type (WT) Kv1.1 activity, as does its prominent metabolite, the abietane diterpenoid pisiferic acid. Uniquely, pisiferic acid also restored function in 12/12 EA1-linked mutant Kv1.1 channels tested in vitro. Crucially, pisiferic acid (1 mg/kg) restored WT function in Kv1.1E283K/+ mice, a model of human EA1. Experimentally validated all-atom molecular dynamics simulations in a neuron-like membrane revealed that the Kv1.1 voltage-sensing domain (VSD) also acts as a ligand-binding domain akin to those of classic ligand-gated channels; binding of pisiferic acid induces a conformational shift in the VSD that ligand-dependently opens the pore. Conifer metabolite pisiferic acid is a promising and versatile therapeutic lead for EA1 and other Kv1.1-linked disorders.

Methods for calculating the relative binding free energy (RBFE) between ligands to a target protein are gaining importance in the structure-based drug discovery domain, especially as methodological advances and automation improve accuracy and ease of use. In an RBFE calculation, the difference between the binding affinities of two ligands to a protein is calculated by transforming one ligand into another, in the protein-ligand complex, and in solvent. Alchemical binding free energy calculations are often used for such ligand transformations. Such calculations are not without challenges, however; for example, it can be challenging to handle interfacial waters when these play a crucial role in mediating protein-ligand binding. In some cases, the exchange of the interfacial waters with solvent water might be very infrequent in the course of typical molecular simulations, and such interfacial waters can be considered trapped on the simulation time scale. In these cases, RBFE calculation between two ligands, where one ligand binds with a trapped water while the other ligand displaces it, can result in inaccuracies if the surrounding water structure is not sampled adequately for both ligands. So far, a popular choice for treating the trapped waters in RBFE calculations is to combine free energy calculations with enhanced sampling methods that insert/delete waters in the binding site. Despite recent developments in the enhanced sampling methods, they can result in hysteresis in the RBFE estimate, depending on whether the simulations were started with or without the trapped waters. In this study, we introduce an alternative method, separation of states, to calculate the RBFE between ligand pairs where the ligands bind to the protein with different numbers/positions of trapped waters. The separation of states approach treats the sampling of the trapped waters separately from the free energy calculation of the ligand transformation. In our method, a trapped water in proteins binding site is decoupled from the system first, and the cavity created by its decoupling is stabilized. We then grow a larger ligand into this cavity- a ligand that is known to displace the trapped water. In this study, we show that our method results in precise and accurate estimates of RBFEs for ligand pairs involving the rearrangement of trapped water via RBFE calculations for five such ligand pairs. We have optimized our simulation protocol to be suited for large distributed computational resources and have automated our RBFE calculation workflow.

RNA sequences encode structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but a complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA in native environments. Existing tools rely primarily on electrophiles that are constitutively on or triggered by UV light, often resulting in high background. Here we describe an alternative, chemically triggered approach to cross-link RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes─electrophiles that can trap neighboring nucleophiles to forge covalent cross-links. As a proof-of-concept, we conjugated a CpO motif to thiazole orange (TO-1). TO-1-CpO bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, as confirmed by fluorescence turn-on. After phosphine administration, covalent cross-links were formed between the CpO and RNA. Cross-linking was both time and dose dependent. We further applied the chemically triggered tools to model RNAs under biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

The assembly of the β-amyloid peptide Aβ into toxic oligomers plays a significant role in the neurodegeneration associated with the pathogenesis of Alzheimers disease. Our laboratory has developed N-methylation as a tool to enable X-ray crystallographic studies of oligomers formed by macrocyclic β-hairpin peptides derived from Aβ. In this investigation, we set out to determine whether α-methylation could be used as an alternative to N-methylation in studying the oligomerization of a β-hairpin peptide derived from Aβ. α-Methylation permits the crystallographic assembly of a triangular trimer and ball-shaped dodecamer, resembling assemblies formed by the N-methylated homolog. Subtle differences are observed in the conformation of the α-methylated peptide when compared to the N-methylated homolog. Notably, α-methylation appears to promote a flatter and more extended β-sheet conformation than that of N-methylated β-sheets or a typical unmodified β-sheet. α-Methylation provides an alternative to N-methylation in X-ray crystallographic studies of oligomers formed by peptides derived from Aβ, with the attractive feature of preserving NH hydrogen-bond donors along the peptide backbone.

Two-pore domain, outwardly rectifying potassium (TOK) channels are exclusively expressed in fungi. Human fungal pathogen TOK channels are potential antifungal targets, but TOK channel modulation in general is poorly understood. Here, we discovered that Candida albicans TOK (CaTOK) is regulated by extracellular pH, in contrast to TOK channels from other fungal species tested. Low pH increased CaTOK channel outward currents (pKa = 6.0), hyperpolarized the voltage-dependence of TOK activation, and increased pore selectivity for K⁺ over Na⁺, shifting the reversal potential (E _REV) toward E _K. Mutating H144 in the S1-S2 extracellular linker partially diminished pH sensitivity, suggesting H144 forms part of the CaTOK pH sensor. Functional analysis of chimeras made with pH-insensitive Saccharomyces cerevisiae TOK and point mutants revealed that CaTOK V462 and S466 in the final transmembrane segment complete the pH-responsive elements. A tripartite network of residues thus endows CaTOK with the ability to respond functionally to changes in pH.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the worlds deadliest infectious disease. Mtb uses a variety of mechanisms to evade the human hosts defenses and survive intracellularly. Mtbs oxidative stress response enables Mtb to survive within activated macrophages, an environment with reactive oxygen species and low pH. Dye-decolorizing peroxidase (DyP), an enzyme involved in Mtbs oxidative stress response, is encapsulated in a nanocompartment, encapsulin (Enc), and is important for Mtbs survival in macrophages. Encs are homologs of viral capsids and encapsulate cargo proteins of diverse function, including those involved in iron storage and stress responses. DyP contains a targeting peptide (TP) at its C-terminus that recognizes and binds to the interior of the Enc nanocompartment. Here, we present the crystal structure of the Mtb-Enc•DyP complex and compare it to cryogenic-electron microscopy (cryo-EM) Mtb-Enc structures. Investigation into the canonical pores formed at symmetrical interfaces reveals that the five-fold pore for the Mtb-Enc crystal structure is strikingly different from that observed in cryo-EM structures. We also observe DyP-TP electron density within the Mtb-Enc shell. Finally, investigation into crystallographic small-molecule binding sites gives insight into potential novel avenues by which substrates could enter Mtb-Enc to react with Mtb-DyP.

Chronic dysregulation of microglial phenotypic balance contributes to prolonged neuroinflammation and neurotoxicity, which is a hallmark of neurodegenerative diseases. Thus, targeting microglial inflammatory signaling represents a promising therapeutic strategy for neurodegenerative diseases. Regulator of G protein Signaling 10 (RGS10) is highly expressed in microglia, where it suppresses pro-inflammatory signaling. However, RGS10 is silenced following microglial activation, augmenting inflammatory responses. While modulating RGS10 expression is a promising strategy to suppress pro-inflammatory microglial activation, no chemical tools with this ability exist. We developed a phenotypic high-throughput assay to screen for compounds with the ability to reverse interferon-γ (IFNγ)-induced RGS10 silencing in BV-2 cells. Identified hits had no effect on RGS10 expression in the absence of stimulus or in response to lipopolysaccharide (LPS). Furthermore, the hits reversed some of the inflammatory gene expression induced by IFNγ. This is the first demonstration of the potential for small molecule intervention to modulate the RGS10 expression in microglia.