A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase-9.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

The Quinazolinone Allosteric Inhibitor of PBP 2a Synergizes with Piperacillin and Tazobactam against Methicillin-Resistant Staphylococcus aureus.

The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA). The quinazolinones target cell wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein 2a (PBP 2a). We investigated the potential for synergism of a lead quinazolinone with several antibiotics of different classes using checkerboard and time-kill assays. The quinazolinone synergized with ß-lactam antibiotics. The combination of the quinazolinone with commercial piperacillin-tazobactam showed bactericidal synergy at sub-MICs of all three drugs. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the ß-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target, PBP 2. Furthermore, the quinazolinone binds to the allosteric site of PBP 2a, triggering the allosteric response. This leads to the opening of the active site, which, in turn, binds another molecule of piperacillin. In other words, PBP 2a, which is not normally inhibited by piperacillin, becomes vulnerable to inhibition in the presence of the quinazolinone. The collective effect is the impairment of cell wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP 2a provide support for the proposed mechanism of action.

Acceleration of diabetic wound healing using a novel protease-anti-protease combination therapy.

Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.

Synergistic, collaterally sensitive ß-lactam combinations suppress resistance in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple ß-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other clinical MRSA isolates in vitro and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem-penicillin-ß-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to ß-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older ß-lactam antibiotics could be effective against MRSA infections in humans.

In Vitro and In Vivo Synergy of the Oxadiazole Class of Antibacterials with ß-Lactams.

The oxadiazole antibacterials target the bacterial cell wall and are bactericidal. We investigated the synergism of ND-421 with the commonly used ß-lactams and non-ß-lactam antibiotics by the checkerboard method and by time-kill assays. ND-421 synergizes well with ß-lactam antibiotics, and it also exhibits a long postantibiotic effect (4.7 h). We also evaluated the in vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant infection model (1.49 log10 bacterial reduction for ND-321 versus 0.36 log10 for linezolid with NRS119) and acts synergistically with ß-lactam antibiotics in vitro and in vivo, and the combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log10 bacterial reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain would have been resistant to oxacillin otherwise.

Phenoxide-Bridged Zinc(II)-Bis(dipicolylamine) Probes for Molecular Imaging of Cell Death.

Cell death is involved in many pathological conditions, and there is a need for clinical and preclinical imaging agents that can target and report cell death. One of the best known biomarkers of cell death is exposure of the anionic phospholipid phosphatidylserine (PS) on the surface of dead and dying cells. Synthetic zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to selectively recognize PS-rich membranes and act as cell death molecular imaging agents. However, there is a need to improve in vivo imaging performance by selectively increasing target affinity and decreasing off-target accumulation. This present study compared the cell death targeting ability of two new deep-red fluorescent probes containing phenoxide-bridged Zn2BDPA complexes. One probe was a bivalent version of the other and associated more strongly with PS-rich liposome membranes. However, the bivalent probe exhibited self-quenching on the membrane surface, so the monovalent version produced brighter micrographs of dead and dying cells in cell culture and also better fluorescence imaging contrast in two living animal models of cell death (rat implanted tumor with necrotic core and mouse thymus atrophy). An (111)In-labeled radiotracer version of the monovalent probe also exhibited selective cell death targeting ability in the mouse thymus atrophy model, with relatively high amounts detected in dead and dying tissue and low off-target accumulation in nonclearance organs. The in vivo biodistribution profile is the most favorable yet reported for a Zn2BDPA complex; thus, the monovalent phenoxide-bridged Zn2BDPA scaffold is a promising candidate for further development as a cell death imaging agent in living subjects.

Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one.

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

Discovery of a new class of non-ß-lactam inhibitors of penicillin-binding proteins with Gram-positive antibacterial activity.

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-ß-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

Enhanced cell death imaging using multivalent zinc(II)-bis(dipicolylamine) fluorescent probes.

There is a clinical need for imaging technologies that can accurately detect cell death in a multitude of pathological conditions. Zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to associate with the anionic phosphatidylserine that is exposed on the surface of dead and dying cells, and fluorescent monovalent Zn2BDPA probes are successful cell death imaging agents. This present study compared the membrane targeting ability of two structurally related deep-red fluorescent probes, bis-Zn2BDPA-SR and tetra-Zn2BDPA-SR, with two and four appended Zn2BDPA units, respectively. Vesicle and cell microscopy studies indicated that a higher number of Zn2BDPA targeting units improved probe selectivity for phosphatidylserine-rich vesicles, and increased probe localization at the plasma membrane of dead and dying cells. The fluorescent probes were also tested in three separate animal models, (1) necrotic prostate tumor rat model, (2) thymus atrophy mouse model, and (3) traumatic brain injury mouse model. In each case, there was more tetra-Zn2BDPA-SR accumulation at the site of cell death than bis-Zn2BDPA-SR. The results indicate that multivalent Zn2BDPA probes are promising molecules for effective imaging of cell death processes in cell culture and in living subjects.

Methylseleninic acid downregulates hypoxia-inducible factor-1α in invasive prostate cancer.

Alternative strategies are needed to control growth of advanced and hormone refractory prostate cancer. In this regard, we investigated the efficacy of methylseleninic acid (MSeA), a penultimate precursor to the highly reactive selenium metabolite, methylselenol, to inhibit growth of invasive and hormone refractory rat (PAIII) and human (PC-3 and PC-3M) prostate cancer cells. Our results demonstrate that MSeA inhibits PAIII cell growth in vitro as well as reduces weights of tumors generated by PAIII cells treated ex vivo. A significant reduction in the number of metastatic lung foci by MSeA treatment was also noted in Lobund-Wistar rats. The PAIII cells along with PC-3, DU145 and PC-3M cells undergo apoptosis after MSeA treatments in both normoxia and hypoxia. Treatment of metastatic rat and human prostate cancer cell lines with MSeA decreased hypoxia-inducible factor-1α (HIF-1α) levels in a dose-dependent manner. Additionally, HIF-1α transcription activity both in normoxic and hypoxic conditions is reduced after MSeA treatment of prostate cancer cells. Furthermore, VEGF and GLUT1, downstream targets of HIF-1α, were also reduced in prostate cancer cells after MSeA treatment. Our study illustrates the efficacy of MSeA in controlling growth of hormone refractory prostate cancer by downregulating HIF-1α, which is possibly occurring through stabilization or increase in prolyl hydroxylase activity.

Proteomics Identification of Targets for Intervention in Pressure Ulcers.

Pressure ulcers (PUs) are chronic wounds that lead to amputations and death. Little is known about why PUs are recalcitrant to healing. Wound healing is mediated by matrix metalloproteinases (MMPs). The 24 MMPs in humans each exist in three forms, of which only one is catalytically competent. We analyzed human PU samples using an affinity resin that exclusively binds to the catalytically competent MMPs. We identified by mass spectrometry the active forms of MMP-1, MMP-8, MMP-9, and MMP-14. Concentrations of MMP-8, MMP-9, and MMP-14 were higher in human PUs compared to the healthy tissue, whereas those for MMP-1 did not change. Decreasing levels of active MMP-9 as the PU improved argued for a detrimental role for this enzyme. In a mouse model of PUs, a highly selective inhibitor for MMP-9 and MMP-14, (R)-ND-336, accelerated wound closure in parallel with significant amelioration of ulcer stage. (R)-ND-336 holds promise as a first-in-class treatment for PUs.

Pro-inflammatory angiogenesis is mediated by p38 MAP kinase.

Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro-inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H2O2, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase- and MMP-9-dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up-regulated ROS production and subsequent endothelial TNF expression. The pro-angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti-angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro-angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti-angiogenic to pro-angiogenic activities in conditions which link oxidative stress and autocrine TNF production.

In vivo targeting of cell death using a synthetic fluorescent molecular probe.

A synthetic, near-infrared, fluorescent probe, named PSS-794 was assessed for its ability to detect cell death in two animal models. The molecular probe contains a zinc(II)-dipicolylamine (Zn(2+)-DPA) affinity ligand that selectively targets exposed phosphatidylserine on the surface of dead and dying cells. The first animal model used rats that were treated with dexamethasone to induce thymic atrophy. Ex vivo fluorescence imaging and histological analysis of excised organs showed thymus uptake of PSS-794 was four times higher than a control fluorophore that lacked the Zn(2+)-DPA affinity ligand. In addition, the presence of PSS-794 produced a delayed and higher build up of dead and dying cells in the rat thymus. The second animal model employed focal beam radiation to induce cell death in tumor-bearing rats. Whole-body and ex vivo imaging showed that the amount of PSS-794 in a radiation-treated tumor was almost twice that in a non-treated tumor. The results indicate that PSS-794 may be useful for preclinical optical detection of tumor cell death due to therapy.

Synthesis and evaluation of 5-lipoxygenase translocation inhibitors from acylnitroso hetero-Diels-Alder cycloadducts.

Acylnitroso cycloadducts have proven to be valuable intermediates in the syntheses of a plethora of biologically active molecules. Recently, organometallic reagents were shown to open bicyclic acylnitroso cycloadducts and, more interestingly, the prospect of highly regioselective openings was raised. This transformation was employed in the synthesis of a compound with excellent inhibitory activity against 5-lipoxygenase ((±)-4a, IC(50) 51 nM), an important mediator of inflammation intimately involved in a number of disease states including asthma and cancer. Optimization of the copper-mediated organometallic ring opening reaction was accomplished allowing the further exploration of the biological activity. Synthesis of a number of derivatives with varying affinity for metal binding as well as pendant groups in a range of sizes was accomplished. Analogues were tested in a whole cell assay which revealed a subset of the compounds to be inhibitors of enzyme translocation, a mode of action not previously known and, potentially, extremely important for better understanding of the enzyme and inhibitor development. Additionally, the lead compound was tested in vivo in an established colon cancer model and showed very encouraging anti-tumorogenic properties.

Selective MMP-9 Inhibitor (R)-ND-336 Alone or in Combination with Linezolid Accelerates Wound Healing in Infected Diabetic Mice.

Diabetic foot ulcers (DFUs) are a common complication of diabetes that are recalcitrant to healing due to persistent inflammation. The majority of DFUs have bacterial biofilms, with Staphylococcus epidermidis as a predominant bacterium, requiring infection control with antibiotics before treatment of the wound. Matrix metalloproteinases (MMPs) play roles in the pathology and repair of DFUs. However, defining the roles of the 24 human MMPs has been challenging due to the presence of three forms for each MMP, of which only one is catalytically competent, and the lack of convenient methods to distinguish among the three forms of MMPs. Using an affinity resin that binds only to the active forms of MMPs, with identification and quantification by mass spectrometry, we found that infected wounds in mice had increased levels of active MMP-9 compared to uninfected ones, paralleling infected human DFUs. MMP-9 activity prevents diabetic wounds from healing. We evaluated the efficacy of the selective small-molecule MMP-9 inhibitor, (R)-ND-336, in the infected diabetic mouse model of wound healing and showed that (R)-ND-336 alone or in combination with the antibiotic linezolid improves wound healing by inhibiting the detrimental MMP-9, mitigating macrophage infiltration to diminish inflammation, and increasing angiogenesis to restore the normal wound healing process. An advantage of this strategy is the ability to administer (R)-ND-336 concurrently with an antibiotic.

Structure-Activity Relationship for the Picolinamide Antibacterials that Selectively Target Clostridioides difficile.

Clostridioides difficile is a leading health threat. This pathogen initiates intestinal infections during gut microbiota dysbiosis caused by oral administration of antibiotics. C. difficile is difficult to eradicate due to its ability to form spores, which are not susceptible to antibiotics. To address the urgent need for treating recurrent C. difficile infection, antibiotics that selectively target C. difficile over common gut microbiota are needed. We herein describe the class of picolinamide antibacterials which show potent and selective activity against C. difficile. The structure-activity relationship of 108 analogues of isonicotinamide 4, a compound that is equally active against methicillin-resistant Staphylococcus aureus and C. difficile, was investigated. Introduction of the picolinamide core as exemplified by analogue 87 resulted in exquisite potency and selectivity against C. difficile. The ability of the picolinamide class to selectively target C. difficile and to prevent gut dysbiosis holds promise for the treatment of recurrent C. difficile infection.

Optical imaging of mammary and prostate tumors in living animals using a synthetic near infrared zinc(II)-dipicolylamine probe for anionic cell surfaces.

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells.

Synthesis and antitumor properties of selenocoxib-1 against rat prostate adenocarcinoma cells.

Hormone refractory prostate cancer poses a huge problem and standard of care chemotherapy has not been very successful. We used a novel strategy to combine properties of 2 well-studied class of compounds (selenium and COX-2 inhibitor) and examined the resulting effectiveness against prostate cancer. Bearing in mind that sulfonamide moiety and pyrazole ring is important for the proapoptotic activity of Celecoxib, we synthesized a selenium derivative, Selenocoxib-1, by modifying Celecoxib at position 3 of the pyrazole ring. The PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat were used to examine the efficacy of Selenocoxib-1 in vitro. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 in vitro. The IC(50) in PAIII and PC-3M cells for Selenocoxib-1 was about 5 microM, while for Celecoxib it was more than 20 microM. Selenocoxib-1 induced apoptosis in a dose-dependent manner in the PAIII cells. COX-2 expression in PAIII cells was downregulated by Celecoxib and Selenocoxib-1 at 20 and 5 microM, respectively; the COX-2 activity was, however, not affected by Selenocoxib-1. Following treatment with Selenocoxib-1, PAIII cells resulted in dose-dependent decrease in HIF-1alpha, p-AKT and Bcl-2 levels. A reduction in weights was observed in subcutaneous tumors produced by PAIII cells pretreated with Selenocoxib-1 as compared to Celecoxib in LW rats. Further, following 1 week Selenocoxib-1 treatment of PAIII tumors resulted in significant reduction of tumor weights. This study demonstrates that Selenocoxib-1 is more effective against prostate cancer than Celecoxib.