Differential effects of inosine monophosphate dehydrogenase (IMPDH/GuaB) inhibition in Acinetobacter baumannii and Escherichia coli.

Inosine 5'-monophosphate dehydrogenase (IMPDH), known as GuaB in bacteria, catalyzes the rate-limiting step in de novo guanine biosynthesis and is conserved from humans to bacteria. We developed a series of potent inhibitors that selectively target GuaB over its human homolog. Here, we show that these GuaB inhibitors are bactericidal, generate phenotypic signatures that are distinct from other antibiotics, and elicit different time-kill kinetics and regulatory responses in two important Gram-negative pathogens: Acinetobacter baumannii and Escherichia coli. Specifically, the GuaB inhibitor G6 rapidly kills A. baumannii but only kills E. coli after 24 h. After exposure to G6, the expression of genes involved in purine biosynthesis and stress responses change in opposite directions while siderophore biosynthesis is downregulated in both species. Our results suggest that different species respond to GuaB inhibition using distinct regulatory programs and possibly explain the different bactericidal kinetics upon GuaB inhibition. The comparison highlights opportunities for developing GuaB inhibitors as novel antibiotics.IMPORTANCEA. baumannii is a priority bacterial pathogen for which development of new antibiotics is urgently needed due to the emergence of multidrug resistance. We recently developed a series of specific inhibitors against GuaB, a bacterial inosine 5'-monophosphate dehydrogenase, and achieved sub-micromolar minimum inhibitory concentrations against A. baumannii. GuaB catalyzes the rate-limiting step of de novo guanine biosynthesis and is highly conserved across bacterial pathogens. This study shows that inhibition of GuaB induced a bacterial morphological profile distinct from that of other classes of antibiotics, highlighting a novel mechanism of action. Moreover, our transcriptomic analysis showed that regulation of de novo purine biosynthesis and stress responses of A. baumannii upon GuaB inhibition differed significantly from that of E. coli.

Discovery of GuaB inhibitors with efficacy against Acinetobacter baumannii infection.

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.

A Comparison of Common Quantum Dot Alternatives to Cadmium-Based Quantum Dots on the Basis of Liver Cytotoxicity.

Fluorescent nanoparticles known as quantum dots (QDs) have unique properties that make them useful in biomedicine. Specifically, CdSe/ZnS QDs, while good at fluorescing, show toxicity. Due to this, safer alternatives have been developed. This study uses a tetrazolium dye (XTT) viability assay, reactive oxygen species (ROS) fluorescent imaging, and apoptosis to investigate the effect of QD alternatives InP/ZnS, CuInS2/ZnS, and nitrogen-doped carbon dots (NCDs) in liver cells. The liver is a possible destination for the accumulation of QDs, making it an appropriate model for testing. A cancerous liver cell line known as HepG2 and an immortalized liver cell line known as THLE-2 were used. At a nanomolar range of 10-150, HepG2 cells demonstrated no reduced cell viability after 24 h. The XTT viability assay demonstrated that CdSe/ZnS and CuInS2/ZnS show reduced cell viability in THLE-2 cells with concentrations between 50 and 150 nM. Furthermore, CdSe/ZnS- and CuInS2/ZnS-treated THLE-2 cells generated ROS as early as 6 h after treatment and elevated apoptosis after 24 h. To further corroborate our results, apoptosis assays revealed an increased percentage of cells in the early stages of apoptosis for CdSe/ZnS-treated (52%) and CuInS2/ZnS-treated (38%) THLE-2. RNA transcriptomics revealed heavy downregulation of cell adhesion pathways such as wnt, cadherin, and integrin in all QDs except NCDs. In conclusion, NCDs show the least toxicity toward these two liver cell lines. While demonstrating less toxicity than CdSe/ZnS, the metallic QDs (InP/ZnS and CuInS2/ZnS) still demonstrate potential concerns in liver cells. This study serves to explore the toxicity of QD alternatives and better understand their cellular interactions.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

The cytotoxic effects of prazosin, chlorpromazine, and haloperidol on hepatocellular carcinoma and immortalized non-tumor liver cells.

Liver cancer annually accounts for over 800,000 cases and 700,000 deaths worldwide. Hepatocellular carcinoma is responsible for over 80% of liver cancer cases. Due to ineffective treatment options and limited surgical interventions, hepatocellular carcinoma is notoriously difficult to treat. Nonetheless, drugs utilized for other medical conditions, such as the antihypertensive medication prazosin, the neuroleptic medication chlorpromazine, and the neuroleptic medication haloperidol, have gained attention for their potential anti-cancer effects. Therefore, this study used these medications for investigating toxicity to hepatocellular carcinoma while testing the adverse effects on a noncancerous liver cell line model THLE-2. After treatment, an XTT cell viability assay, cell apoptosis assay, reactive oxygen species (ROS) assay, apoptotic proteome profile, and western blot were performed. We calculated IC50 values for chlorpromazine and prazosin to have a molar range of 35-65 µM. Our main findings suggest the capability of both of these treatments to reduce cell viability and generate oxidative stress in HepG2 and THLE-2 cells (p value < 0.05). Haloperidol, however, failed to demonstrate any reduction in cell viability revealing no antitumor effect up to 100 µM. Based on our findings, a mechanism of cell death was not able to be established due to lack of cleaved caspase-3 expression. Capable of bypassing many aspects of the lengthy, costly, and difficult cancer drug approval process, chlorpromazine and prazosin deserve further investigation for use in conjunction with traditional chemotherapeutics.

Potent and selective binders of the E3 ubiquitin ligase ZNRF3 stimulate Wnt signaling and intestinal organoid growth.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

Naturally occurring highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b infection in three domestic cats in North America during 2023.

The Eurasian strain of highly pathogenic avian influenza (HPAI) H5N1 is a devastating pathogen for birds that also has the capacity to infect mammals. This report describes the presentation, clinical case findings (including haemogram and serum biochemistry), gross and microscopic lesions and virus detection in three HPAI H5N1-infected domestic cats from the USA in 2023. All three cats presented with neurological abnormalities and were euthanized due to a poor prognosis within 2 days (two cats) or 10 days (one cat) of known clinical disease onset. Necropsy consistently revealed pulmonary congestion and oedema, and cerebrocortical malacia with haemorrhage was also seen in the cat that survived for 10 days. On histology, all cats had necrotizing encephalitis and interstitial pneumonia with pulmonary congestion, oedema, vasculitis and vascular thrombosis. One cat also had microscopic multifocal necrosis in the liver, pancreas and an adrenal gland. To our knowledge, this report is the first to detail pathological findings in HPAI H5N1 naturally-infected cats during the widespread outbreak in North America beginning in 2021, and that describes a cat surviving for 10 days after onset of HPAI H5N1 encephalitis.

Apoptotic pathway protein expression variance in metal oxide and quantum dot treated HeLa cells.

Using the HeLa cell line as a cancerous model, apoptotic protein expression was assessed upon various nanoparticle treatments. Utilizing a known chemotherapeutic agent, cisplatin, as a positive control for induction of apoptosis, several metal oxides (ZnO and CuO) and quantum dots (CdSe/ZnS and InP/ZnS) were investigated for their ability to express apoptotic markers. ZnO, CuO, green CdSe/ZnS, and green InP/ZnS were treated for 24 hours at their IC50 value. Western blot techniques were used to measure protein expression of phosphorylated p53 (ser15), PUMA, and p21 which are involved in signal transduction of apoptosis. CuO, ZnO, and CdSe/ZnS demonstrated considerable p53 activation at 24 hrs compared to the non-treated control. At the IC50 value, CdSe/ZnS quantum dots were the quickest at activating p53 by phosphorylation at the Serine 15 residue. Together, our results provide new insight into the apoptotic mechanism behind these treatments and lead to improved treatments against cancer.

Host-genetic-based outcome of co-infection by PCV2b and PRRSV in pigs.

Replication of porcine circovirus type 2 (PCV2), an important worldwide swine pathogen, has been demonstrated to be influenced by host genotype. Specifically, a missense DNA polymorphism (SYNGR2 p.Arg63Cys) within the SYNGR2 gene was demonstrated to contribute to variation in PCV2b viral load and subsequent immune response following infection. PCV2 is known to induce immunosuppression leading to an increase in susceptibility to subsequent infections with other viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). In order to assess the role of SYNGR2 p.Arg63Cys in co-infections, pigs homozygous for the favorable SYNGR2 p.63Cys (N = 30) and unfavorable SYNGR2 p.63Arg (N = 29) alleles were infected with PCV2b followed a week later by a challenge with PRRSV. A lower PCV2b viremia (P < 0.001) and PCV2-specific IgM antibodies (P < 0.005) were observed in SYNGR2 p.63Cys compared to SYNGR2 p.63Arg genotypes. No significant differences in PRRSV viremia and specific IgG antibodies were observed between SYNGR2 genotypes. Lung histology score, an indicator of disease severity, was lower in the pigs with SYNGR2 p.63Cys genotypes (P < 0.05). Variation in the lung histology scores within SYNGR2 genotypes suggests that additional factors, environmental and/or genetic, could be involved in disease severity.

Porcine circovirus type 2 (PCV2) is an important virus involved in the onset of a group of severe disease symptoms commonly known as porcine circovirus associated diseases (PCVAD). Vaccination options exist for PCV2, though the severity of PCVAD can be influenced by the presence of additional co-infecting pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), for which vaccination is still a challenge. Host genetic resistance is a potential avenue for solving this problem. Previously, a genetic polymorphism in the SYNGR2 gene was found to be associated with PCV2b viremia and immune response. The aim of this study was to determine the impact of this polymorphism in pigs experimentally co-infected with PCV2b and PRRSV. Pigs were weighed, and blood was collected at various days following infection to measure viremia and antibodies. Histological analysis was performed at the experiment completion to assess disease severity in lungs and lymph nodes. The results showed that variation within the SYNGR2 gene is involved in PCV2b disease progression including lung histology scores, but no evidence was seen in response to PRRSV infection.

Chemical space docking enables large-scale structure-based virtual screening to discover ROCK1 kinase inhibitors.

With the ever-increasing number of synthesis-on-demand compounds for drug lead discovery, there is a great need for efficient search technologies. We present the successful application of a virtual screening method that combines two advances: (1) it avoids full library enumeration (2) products are evaluated by molecular docking, leveraging protein structural information. Crucially, these advances enable a structure-based technique that can efficiently explore libraries with billions of molecules and beyond. We apply this method to identify inhibitors of ROCK1 from almost one billion commercially available compounds. Out of 69 purchased compounds, 27 (39%) have Ki values < 10 µM. X-ray structures of two leads confirm their docked poses. This approach to docking scales roughly with the number of reagents that span a chemical space and is therefore multiple orders of magnitude faster than traditional docking.

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Antibody interfaces revealed through structural mining.

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody fragment antigen-binding (Fab) regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, ß-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.

Testing oxygenated microbubbles via intraperitoneal and intrathoracic routes on a large pig model of LPS-induced acute respiratory distress syndrome.

With a mortality rate of 46% before the onset of COVID-19, acute respiratory distress syndrome (ARDS) affected 200,000 people in the US, causing 75,000 deaths. Mortality rates in COVID-19 ARDS patients are currently at 39%. Extrapulmonary support for ARDS aims to supplement mechanical ventilation by providing life-sustaining oxygen to the patient. A new rapid-onset, human-sized pig ARDS model in a porcine intensive care unit (ICU) was developed. The pigs were nebulized intratracheally with a high dose (4 mg/kg) of the endotoxin lipopolysaccharide (LPS) over a 2 h duration to induce rapid-onset moderate-to-severe ARDS. They were then catheterized to monitor vitals and to evaluate the therapeutic effect of oxygenated microbubble (OMB) therapy delivered by intrathoracic (IT) or intraperitoneal (IP) administration. Post-LPS administration, the PaO2 value dropped below 70 mmHg, the PaO2 /FiO2 ratio dropped below 200 mmHg, and the heart rate increased, indicating rapidly developing (within 4 h) moderate-to-severe ARDS with tachycardia. The SpO2 and PaO2 of these LPS-injured pigs did not show significant improvement after OMB administration, as they did in our previous studies of the therapy on small animal models of ARDS injury. Furthermore, pigs receiving OMB or saline infusions had slightly lower survival than their ARDS counterparts. The OMB administration did not induce a statistically significant or clinically relevant therapeutic effect in this model; instead, both saline and OMB infusion appeared to lower survival rates slightly. This result is significant because it contradicts positive results from our previous small animal studies and places a limit on the efficacy of such treatments for larger animals under more severe respiratory distress. While OMB did not prove efficacious in this rapid-onset ARDS pig model, it may retain potential as a novel therapy for the usual presentation of ARDS in humans, which develops and progresses over days to weeks.

In situ printing of scaffolds for reconstruction of bone defects.

Bone defects are commonly caused by traumatic injuries and tumor removal and critically sized defects overwhelm the regenerative capacity of the native tissue. Reparative strategies such as auto, xeno, and allografts have proven to be insufficient to reconstruct and regenerate these defects. For the first time, we introduce the use of handheld melt spun three dimensional printers that can deposit materials directly within the defect site to properly fill the cavity and form free-standing scaffolds. Engineered composite filaments were generated from poly(caprolactone) (PCL) doped with zinc oxide nanoparticles and hydroxyapatite microparticles. The use of PCL-based materials allowed low-temperature printing to avoid overheating of the surrounding tissues. The in situ printed scaffolds showed moderate adhesion to wet bone tissue, which can prevent scaffold dislocation. The printed scaffolds showed to be osteoconductive and supported the osteodifferentiation of mesenchymal stem cells. Biocompatibility of the scaffolds upon in vivo printing subcutaneously in mice showed promising results. STATEMENT OF SIGNIFICANCE.

Miniaturized Needle Array-Mediated Drug Delivery Accelerates Wound Healing.

A major impediment preventing normal wound healing is insufficient vascularization, which causes hypoxia, poor metabolic support, and dysregulated physiological responses to injury. To combat this, the delivery of angiogenic factors, such as vascular endothelial growth factor (VEGF), has been shown to provide modest improvement in wound healing. Here, the importance of specialty delivery systems is explored in controlling wound bed drug distribution and consequently improving healing rate and quality. Two intradermal drug delivery systems, miniaturized needle arrays (MNAs) and liquid jet injectors (LJIs), are evaluated to compare effective VEGF delivery into the wound bed. The administered drug's penetration depth and distribution in tissue are significantly different between the two technologies. These systems' capability for efficient drug delivery is first confirmed in vitro and then assessed in vivo. While topical administration of VEGF shows limited effectiveness, intradermal delivery of VEGF in a diabetic murine model accelerates wound healing. To evaluate the translational feasibility of the strategy, the benefits of VEGF delivery using MNAs are assessed in a porcine model. The results demonstrate enhanced angiogenesis, reduced wound contraction, and increased regeneration. These findings show the importance of both therapeutics and delivery strategy in wound healing.

Identification of Noncompetitive Protein-Ligand Interactions for Structural Optimization.

For efficient structure-guided drug design, it is important to have an excellent understanding of the quality of interactions between the target receptor and bound ligands. Identification and characterization of poor intermolecular contacts offers the possibility to focus design efforts directly on ligand regions with suboptimal molecular recognition. To enable a more straightforward identification of these in a structural model, we use a suitably enhanced version of our previously introduced statistical ratio of frequencies (RF) approach. This allows us to highlight protein-ligand interactions and geometries that occur much less often in the Protein Data Bank than would be expected from the exposed surface areas of the interacting atoms. We provide a comprehensive overview of such noncompetitive interactions and geometries for a set of common ligand substituents. Through retrospective case studies on congeneric series and single-point mutations for several pharmaceutical targets, we illustrate how knowledge of noncompetitive interactions could be exploited in the drug design process.

Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors.

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.