Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) remain among the most prevalent human pathogens, imposing a substantial global health burden. Recurrent orolabial and genital lesions affect millions worldwide, reflecting lifelong infection and the limited ability of current therapies to prevent reactivation and transmission. There have been no major therapeutic advances in over 40 years, and an effective vaccine remains elusive. New antivirals with novel mechanisms are needed, both to combat resistant strains and to enable combination therapies that reduce toxicity and potentially limit persistence. Recombination is a universal process essential for DNA replication, repair, and genome diversification. Increasing evidence indicates that HSV replication involves single-strand annealing (SSA) mediated by a conserved two-component recombination system composed of UL12, a 5'→3' exonuclease, and ICP8, the viral SSAP (single-strand annealing protein). Both UL12 nuclease activity and ICP8 DNA binding and annealing functions are required for productive infection, identifying them as promising targets for broad-spectrum antiviral development across human herpesviruses.

The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8Δ60) bound to poly(dT)25 or poly(dA)25 ssDNA at 3.0 to 3.1 Å resolution, along with higher-resolution apo structures of surface-entropy-reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, π-stacking and phosphate backbone interactions. In the poly(dT)25 complex, coordination of a Zn2+ ion stabilizes the zinc finger motif; whereas, in the poly(dA)25 complex, Zn2+ displacement promotes disulfide bond formation that effectively locks the protein into an altered conformation. Microscale thermophoresis and label-free differential scanning fluorimetry reveal a strong preference for pyrimidine-rich sequences, with nanomolar affinity for poly(dT)25 and micromolar for poly(dA)25. Structural modeling identified Y543, R576, R772, R793, Y988, and F998 as key DNA-contact residues. Alanine substitutions caused severe replication defects, particularly for R772A, Y988A, and F998A. ssDNA binding induces ∼26 Å displacement and ∼35 degree rotation of the C-terminal domain and ordering of flexible loops, suggesting a mechanism for cooperative filament assembly. These structures define the molecular determinants of ICP8-ssDNA recognition, reveal thymidine bias and provide a framework for targeting ICP8-mediated functions in herpesvirus replication.

Herpes simplex virus-1 (HSV-1) UL12 gene encodes a well-conserved 5' → 3' alkaline exonuclease. UL12 collaborates with the HSV single-strand DNA binding protein ICP8 to mediate recombination-dependent replication of viral DNA and is essential for the production of DNA that can be packaged into infectious virus. The UL12 gene has orthologs in the eight other human herpesviruses, including UL98 in HCMV and SOX in KSHV, which are also essential for virus production. We have developed viral nuclease inhibitors (VNIs) of HSV-1 UL12 that potently block its nuclease activity and display strong antiviral effects in cell culture. These inhibitors are also effective against alkaline nucleases from the β-HHV HCMV (UL98) and the γ-HHV KSHV (SOX), and we have demonstrated antiviral activity against HSV-1 and HCMV in cell culture. In this work, we describe the first crystal structure of an alphaherpesvirus alkaline nuclease (UL12.5), which was used to elucidate structure activity relationships and improve the selectivity of our inhibitors. These VNIs exhibit EC50 and IC50 values in the nanomolar to low micromolar range. Our findings highlight the potential of targeting HHV alkaline nucleases with novel small molecules, paving the way for the development of new therapies that can be broadly antiviral on their own or in combination with nucleoside analogs.

Herpes simplex virus (HSV) replicates by forming DNA concatemers using a single strand annealing mechanism mediated by the multifunctional HSV protein ICP8. ICP8 binds cooperatively to form nucleoprotein filaments on ssDNA and promotes annealing of complementary single strands. These functions are believed to be mediated by ICP8:ICP8 protein-protein interactions (PPIs) between the flexible C-terminal domain of ICP8 and the N-terminal domain of a second ICP8 monomer. We previously presented genetic evidence for an ICP8:ICP8 PPI hotspot between two hydrophobic regions and showed that it is essential for the cooperative binding of ICP8 to ssDNA, ICP8 filament formation in vitro, and viral replication. This involves interactions between F1142, N1143, and F1144 (FNF) in the C terminal disordered tail of ICP8 and the hydrophobic residues (F843 and W844) in the head region of the N-terminal domain on a second molecule of ICP8. In this article, we propose an additional second distinct PPI hotspot involving residues R922, within the body region of the N-terminal domain of ICP8, and D1087, within the C-terminal domain (CTD). Biophysical studies using thermal melting assays and microscale thermophoresis support an interaction between the ICP8-CTD and the N-terminal domain of ICP8 and show that mutations within each of these hotspots disrupt the interactions. Furthermore, we demonstrate that a construct consisting of the ICP8-CTD can antagonize the assembly of ICP8 filaments in vitro, block viral replication compartment formation and virus production, suggesting that these PPI hotspots may be useful in the development of new antiviral therapies.

Antimicrobial resistance (AMR) to existing antibiotics poses a critical global health challenge, with significant morbidity and mortality from bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains (VISA, VRSA) are among the most pressing threats, particularly for vulnerable populations. To combat this crisis, the development of novel therapeutic strategies is imperative. We report the pharmacokinetic evaluation of a promising class of propargyl-linked diaminopyrimidine dihydrofolate reductase (DHFR) inhibitors with potent activity against drug-resistant bacteria, including MRSA and VISA strains. Previous studies have demonstrated minimum inhibitory concentration (MIC) values below 1 μg.mL-1 for several compounds in this series. Here, we detail the development and validation of an LC-QQQ bioanalytical method for seven propargyl-linked diaminopyrimidine analogues. Pharmacokinetic studies in a murine model across intravenous (IV), intraperitoneal (IP), and oral (PO) routes revealed substantial variability in parameters such as half-life (t₁/₂), area under the curve (AUC), and peak plasma concentration (Cmax). Compound 38C1 demonstrated favorable solubility, a higher maximum tolerated dose, and oral bioavailability of 20 %, making it a lead candidate. Pharmacokinetic-to-MIC ratio analyses showed that 38C1 maintained plasma concentrations significantly above MIC values for multiple S. aureus strains, including MRSA and VISA. These findings highlight 38C1 as a promising antifolate candidate for further development. Ongoing studies will assess its efficacy in infection models and refine delivery strategies to maximize therapeutic potential while mitigating resistance development.

Cellular resistance can limit the effectiveness of antifolate drugs for the treatment of cancer and autoimmune diseases. We examined the biochemical and cellular effects of a propargyl linked, non-classical antifolate UCP1162 that shows exceptional potency and resilience in the background of methotrexate resistance. UCP1162 inhibited the human DHFR enzyme with affinity and kinetics comparable to methotrexate (MTX). UCP1162 also inhibited cancer cell proliferation and bound cellular DHFR at low nanomolar concentrations. Leucovorin suppressed the cellular effects of UCP1162, consistent with UCP1162 working as an antifolate. Like other antifolates, UCP1162 reduced acute inflammation in mice and inhibited FLS cell growth and motility. Single cell RNA-seq showed that MTX and UCP1162 generated overlapping gene expression changes after a 48-hour exposure. However, while leukemia cells (CCRF-CEM) resistant to MTX could be readily selected, UCP1162-resistant cells could not be obtained. Long-term exposure to UCP1162 resulted in static culture expressing stem cell genes (CD34, ABCG2, ABCB1), adaptive genes (TCN2, CDKN1A), and genes that might serve as therapeutic targets (TPBG/5T4, TNFRSF10A, ACE). These findings suggest that UCP1162 is a unique tool for studying cellular responses to long-term antifolate treatment and holds promise as a lead compound capable of overcoming some forms of antifolate resistance.

S1: Results from AK306 testing on the NCI60 cancer cell line panel. Testing was done by the National Institutes of Health Developmental Therapeutics Program. S2: AK3-BODIPY arrests YAMC cells in mitosis at 600 and 800 nM. YAMCs were treated with compound overnight and then assayed for mitotic arrest by staining for phospho-histone H3. S3: Tumor-bearing ApcÃŽâ€14/+ mice were treated with five injections of AK306 (30 mg/kg) over the course of three days. Animals were sacrificed and tissue analyzed for PCNA staining (A) and phospho-histone H3 staining (B). Representative staining images are shown in the right panels, with quantified staining shown in the left panels.

Background: High mobility group proteins 1 and 2 (HMGB1 and HMGB2) are 80% conserved in amino acid sequence. The function of HMGB1 in inflammation and fibrosis has been extensively characterized. However, an unaddressed central question is the role of HMGB2 on liver fibrosis. In this study, we provided convincing evidence that the HMGB2 expression was significantly upregulated in human liver fibrosis and cirrhosis, as well as in several mouse liver fibrosis models. Methods: The carbon tetrachloride (CCl 4 ) induced liver fibrosis mouse model was used. AAV8-Hmgb2 was utilized to overexpress Hmgb2 in the liver, while Hmgb2−/− mice were used for loss of function experiments. The HMGB2 inhibitor inflachromene and liposome-shHMGB2 (lipo-shHMGB2) were employed for therapeutic intervention. Results: The serum HMGB2 levels were also markedly elevated in patients with liver fibrosis and cirrhosis. Deletion of Hmgb2 in Hmgb2−/− mice or inhibition of HMGB2 in mice using a small molecule ICM slowed the progression of CCl 4 -induced liver fibrosis despite constant HMGB1 expression. In contrast, AAV8-mediated overexpression of Hmgb2 enchanced CCl 4 -incuded liver fibrosis. Primary hepatic stellate cells (HSCs) isolated from Hmgb2−/− mice showed significantly impaired transdifferentiation and diminished activation of α-SMA, despite a modest induction of HMGB1 protein. RNA-seq analysis revealed the induction of top 45 CCl 4 -activated genes in multiple signaling pathways including integrin signaling and inflammation. The activation of these genes by CCl 4 were abolished in Hmgb2−/− mice or in ICM-treated mice. These included C-X3-C motif chemokine receptor 1 (Cx3cr1) associated with inflammation, cyclin B (Ccnb) associated with cell cycle, DNA topoisomerase 2-alpha (Top2a) associated with intracellular component, and fibrillin (Fbn) and fibromodulin (Fmod) associated with extracellular matrix. Conclusion: We conclude that HMGB2 is indispensable for stellate cell activation. Therefore, HMGB2 may serve as a potential therapeutic target to prevent HSC activation during chronic liver injury. The blood HMGB2 level may also serve as a potential diagnostic marker to detect early stage of liver fibrosis and cirrhosis in humans.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

In this review, we explore recombination in two very different virus families that have become major threats to human health. The Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Coronaviridae are positive-strand RNA viruses (CoVs) that have also become major threats to global health and economic stability, especially in the last two decades. Despite many differences, such as the make-up of their genetic material (DNA vs. RNA) and overall mechanisms of genome replication, both human herpes viruses (HHVs) and CoVs have evolved to rely heavily on recombination for viral genome replication, adaptation to new hosts and evasion of host immune regulation. In this review, we will focus on the roles of three viral exonucleases: two HHV exonucleases (alkaline nuclease and PolExo) and one CoV exonuclease (ExoN). We will review the roles of these three nucleases in their respective life cycles and discuss the state of drug discovery efforts against these targets.

Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs’ inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs’ inhibition, while other PLAs remain unaffected by this resistance mechanism.

Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.

The majority of drug discovery efforts against herpesviruses have focused on nucleoside analogs that target viral DNA polymerases, agents that are associated with dose-limiting toxicity and/or a narrow spectrum of activity. We are pursuing a strategy based on targeting two-metal ion-dependent (TMID) viral enzymes. This family of enzymes consists of structurally related proteins that share common active sites containing conserved carboxylates predicted to coordinate divalent cations essential for catalysis. Compounds that target TMID enzymes, such as HIV integrase and influenza endoribonuclease, have been successfully developed for clinical use. HIV integrase inhibitors have been reported to inhibit replication of herpes simplex virus (HSV) and other herpesviruses; however, the molecular targets of their antiviral activities have not been identified. We employed a candidate-based approach utilizing several two-metal-directed chemotypes and the potential viral TMID enzymatic targets in an effort to correlate target-based activity with antiviral potency. The panel of compounds tested included integrase inhibitors, the anti-influenza agent baloxavir, three natural products previously shown to exhibit anti-HSV activity, and two 8-hydroxyquinolines (8-HQs), AK-157 and AK-166, from our in-house program. The integrase inhibitors exhibited weak overall anti-HSV-1 activity, while the 8-HQs were shown to inhibit both HSV-1 and cytomegalovirus (CMV). Target-based analysis demonstrated that none of the antiviral compounds acted by inhibiting ICP8, contradicting previous reports. On the other hand, baloxavir inhibited the proofreading exonuclease of HSV polymerase, while AK-157 and AK-166 inhibited the alkaline exonuclease UL12. In addition, AK-157 also inhibited the catalytic activity of the HSV polymerase, which provides an opportunity to potentially develop dual-targeting agents against herpesviruses. IMPORTANCE Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance. New therapies with novel modes of action would be important not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities and potentially eliminate infection. Since many essential HHV proteins are well conserved, inhibitors of novel targets would ideally exhibit broad-spectrum activity against multiple HHVs.

Oligodendrocyte precursor cells (OPCs), also known as NG2 cells or polydendrocytes, are distributed widely throughout the developing and mature central nervous system. They remain proliferative throughout life and are an important source of myelinating cells in normal and demyelinating brain as well as a source of glioma, the most common type of primary brain tumor with a poor prognosis. OPC proliferation is dependent on signaling mediated by platelet‐derived growth factor (PDGF) AA binding to its alpha receptor (PDGFRα). Here, we describe a group of structurally related compounds characterized by the presence of a basic guanidine group appended to an aromatic core that is effective in specifically repressing the transcription of Pdgfra but not the related beta receptor (Pdgfrb) in OPCs. These compounds specifically and dramatically reduced proliferation of OPCs but not that of astrocytes and did not affect signal transduction by PDGFRα. These findings suggest that the compounds could be further developed for potential use in combinatorial treatment strategies for neoplasms with dysregulated PDGFRα function.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora , was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Many small molecule natural products with are adorned with a carbohydrate as part of their molecular structure that acts to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. Here we report a new approach for the synthesis of glycosylated small molecule libraries that puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. A number of desosaminyl glycosides were prepared from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.

Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that are of interest due to their cytotoxic properties. MO-OH-Nap is a novel α-substituted tropolone that induces caspase cleavage and upregulates markers associated with the unfolded protein response (UPR) in multiple myeloma (MM) cells. Given previous reports that tropolones may function as iron chelators, we investigated the effects of MO-OH-Nap, as well as the known iron chelator deferoxamine (DFO), in MM cells in the presence or absence of supplemental iron. The ability of MO-OH-Nap to induce apoptosis and upregulate markers of the UPR could be completely prevented by co-incubation with either ferric chloride or ammonium ferrous sulfate. Iron also completely prevented the decrease in BrdU incorporation induced by either DFO or MO-OH-Nap. Ferrozine assays demonstrated that MO-OH-Nap directly chelates iron. Furthermore, MO-OH-Nap upregulates cell surface expression and mRNA levels of transferrin receptor. In vivo studies demonstrate increased Prussian blue staining in hepatosplenic macrophages in MO-OH-Nap-treated mice. These studies demonstrate that MO-OH-Nap-induced cytotoxic effects in MM cells are dependent on the tropolone’s ability to alter cellular iron availability and establish new connections between iron homeostasis and the UPR in MM.