A Potent and Narrow-Spectrum Antibacterial against Clostridioides difficile Infection.

Clostridioides difficile is an anaerobic Gram-positive bacterium that colonizes the gut of patients treated with broad-spectrum antibiotics. The normal gut microflora prevents C. difficile colonization; however, dysbiosis by treatment with broad-spectrum antibiotics causes recurrent C. difficile infection (CDI) in 25% of patients. There are no fully effective antibiotics for multiple recurrent CDIs. We report herein that oxadiazole antibiotics exhibit bactericidal activity against C. difficile vegetative cells. We screened a library of 75 oxadiazoles against C. difficile ATCC 43255. The findings from this collection served as the basis for the syntheses of an additional 58 analogs, which were tested against the same strain. We report a potent (MIC50 = 0.5 µg/mL and MIC90 = 1 µg/mL values for 101 C. difficile strains) and narrow-spectrum oxadiazole (3-(4-(cyclopentyloxy)phenyl)-5-(4-nitro-1H-imidazol-2-yl)-1,2,4-oxadiazole; compound 57), which is not active against common gut bacteria or other tested organisms. Compound 57 is selectively bactericidal against C. difficile and targets cell-wall synthesis.

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.

Dimeric 2-aminoimidazoles are highly active adjuvants for gram-positive selective antibiotics against Acinetobacter baumannii.

The Centers for Disease Control and Prevention (CDC) reports that hospital acquired infections have increased by 65% since 2019. One of the main contributors is the gram-negative bacterium Acinetobacter baumannii. Previously, we reported aryl 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide antibiotics against A. baumannii. Macrolide antibiotics are typically used to treat infections caused by gram-positive bacteria, but are ineffective against most gram-negative bacteria. We describe a new class of dimeric 2-AIs that are highly active macrolide adjuvants, with lead compounds lowering minimum inhibitory concentrations (MICs) to or below the gram-positive breakpoint level against A. baumannii. The parent dimer lowers the clarithromycin (CLR) MIC against A. baumannii 5075 from 32 µg/mL to 1 µg/mL at 7.5 µM (3.4 µg/mL), and a subsequent structure activity relationship (SAR) study identified several compounds with increased activity. The lead compound lowers the CLR MIC to 2 µg/mL at 1.5 µM (0.72 µg/mL), far exceeding the activity of both the parent dimer and the previous lead aryl 2-AI. Furthermore, these dimeric 2-AIs exhibit considerably reduced mammalian cell toxicity compared to aryl-2AI adjuvants, with IC50s of the two lead compounds against HepG2 cells of >200 µg/mL, giving therapeutic indices of >250.

Synthesis of Muramyl-δ-Lactam in Spore Peptidoglycan of Clostridioides difficile.

Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.

Matrix metalloproteinase profiling and their roles in disease.

Matrix metalloproteinases (MMPs) play roles in remodelling of the extracellular matrix that occurs during morphogenesis, repair, and angiogenesis. Dysregulation of extracellular matrix remodelling can lead to cell proliferation, invasion, and tissue fibrosis. Identification of a specific MMP(s) in a disease has been challenging due to the presence of 24 closely-related human MMPs, each existing in three forms, of which only one is active and capable of catalysis. This review focuses on methods for MMP profiling, with particular emphasis on the batimastat affinity resin that binds only to the active forms of MMPs and related ADAMs (a disintegrin and metalloproteinases), which are then identified by mass spectrometry. Use of the batimastat affinity resin has identified targets for intervention in several human diseases.

Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

Background: The median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies. Methods: Public microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance. Results: MT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult. Conclusions: MT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.

Matrix Metalloproteinase-14 as an Instigator of Fibrosis in Human Pterygium and Its Pharmacological Intervention.

There exists a paucity of information on the pathogenesis of pterygium, a benign ocular tumor that scars the cornea and can lead to vision loss. The main recourse for pterygium is surgery; however, recurrence is observed. Matrix metalloproteinases (MMPs) are involved in the pathology of pterygium. The determination of the specific MMP involved among the 24 human enzymes has not been established due to challenges in MMP profiling. We used an affinity resin that binds specifically to the active forms of MMPs in the complex mixture of the cellular proteome. The proteomics analysis identified active MMP-14 and three related metalloproteinases, ADAM9, ADAM10, and ADAM17, in human pterygia. Inhibition of MMP-14 with the small-molecule inhibitor (R)-ND-336 was assessed in cell migration and collagen contraction assays. (R)-ND-336 attenuated human conjunctiva fibroblast migration and mitigated collagen contraction, both activities required for the formation of pterygium. (R)-ND-336 holds the promise of a therapeutic recourse for pterygium as an orphan disease.

MMP-1 and ADAM10 as Targets for Therapeutic Intervention in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF), a fatal disease characterized by excessive matrix degradation and fibrosis, destroys the lung architecture and results in the inability of the lungs to absorb oxygen. The cause(s) of IPF is unknown and current treatments are palliative. Matrix metalloproteinases (MMPs) and A Disintegrin And Metalloproteinases (ADAMs) likely play roles in IPF progression. However, specific MMPs and ADAMs in IPF have not been identified due to challenges in MMP/ADAM profiling. We employed a designer affinity resin that binds exclusively to the active forms of MMPs and ADAMs and found by mass spectrometry higher levels of active MMP-1, ADAM9, ADAM10, and ADAM17 in lung tissues of IPF patients. Inhibition of MMP-1 and ADAM10 with the small-molecule inhibitor GI254023X in an in vitro lung fibrosis assay decreased the profibrotic protein α-smooth muscle actin (α-SMA). Our results indicate that inhibition of MMP-1 and ADAM10 may hold promise in treatment of IPF.

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.

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery.

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with ß-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

Production of Proteins of the SARS-CoV-2 Proteome for Drug Discovery.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19). Its genome encodes two open reading frames for two large proteins, PP1a and PP1ab. Within the two polypeptide stretches, there are two proteases that process the large proteins into 15 discrete proteins essential for the assembly of the virion during its replication. We describe herein the cloning of the genes for these discrete proteins optimized for expression in Escherichia coli, production of the proteins, and their purification to homogeneity. These included all but six: NSP6, which possesses eight transmembrane regions, and five that are small proteins/peptides (E, ORF3b, ORF6, ORF7b, and ORF10). These proteins are intended for experimental validation of small-molecule binders as molecular template hits. The proof of concept was established with the ADP-ribosylhydrolase (ARH) domain of NSP3 in discovery of small-molecule templates that could serve as the basis for further optimization. The hit molecules include one submicromolar and a few low-micromolar binders to the ARH domain. Availability of these proteins in soluble forms opens up the opportunity for discoveries of novel templates with the potential for anti-COVID-19 pharmaceuticals.

Metabolism of the Selective Matrix Metalloproteinase-9 Inhibitor (R)-ND-336.

(R)-ND-336-designated as compound (R)-5-is a highly selective inhibitor of matrix metalloproteinase (MMP)-9 with efficacy in accelerating diabetic wound healing in murine models. (R)-ND-336 belongs to the class of thiirane inhibitors of MMPs and it is currently undergoing Investigation New Drug (IND)-enabling studies. We investigated the in vitro metabolism of (R)-ND-336 using S9 fractions obtained from mice, rats, dogs, minipigs, monkeys, and humans in order to select the rodent and nonrodent species for toxicology studies. Three metabolites were observed. One metabolite, M3, was observed across all species. Metabolite M2 was found in rats, monkeys, and humans. Metabolite M1 was observed only in rats. The identities of the metabolites were suggested by liquid chromatography/tandem mass spectroscopy (LC/MS-MS) analyses, which were authenticated by comparison to synthetic samples. Metabolites M2 and M3 arise from oxidative deamination of (R)-ND-336 by monoamine oxidase to give the arylaldehyde as a transient (and unobserved) intermediate. Reductive metabolism of this aldehyde gives the alcohol metabolite M2, while further oxidative metabolism of the aldehyde produces the carboxylate metabolite M3. A minor route of metabolism, seen only in rats, is N-acetylation of (R)-ND-336 to give the acetamide M1. The metabolism of (R)-ND-336 is distinctly different from that of the prototype member of this thiirane class ((±)-1, lacking the 4-aminomethyl aryl substituent) which is metabolized primarily by oxidation α to the sulfone to lead to a benzenesulfinate metabolite. All three metabolites are poorer MMP-9 inhibitors, compared to (R)-ND-336 (MMP-9, K i = 19 nM): M3, MMP-9 IC50 > 100 µM; M2, K i = 390 nM; and M1, IC50 > 100 µM). The rat and the minipig were selected as the rodent and nonrodent species, respectively, for toxicology studies.

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.

Blood-brain barrier resealing in neuromyelitis optica occurs independently of astrocyte regeneration.

Approximately 80% of neuromyelitis optica spectrum disorder (NMOSD) patients harbor serum anti-aquaporin-4 autoantibodies targeting astrocytes in the CNS. Crucial for NMOSD lesion initiation is disruption of the blood-brain barrier (BBB), which allows the entrance of Abs and serum complement into the CNS and which is a target for new NMOSD therapies. Astrocytes have important functions in BBB maintenance; however, the influence of their loss and the role of immune cell infiltration on BBB permeability in NMOSD have not yet been investigated. Using an experimental model of targeted NMOSD lesions in rats, we demonstrate that astrocyte destruction coincides with a transient disruption of the BBB and a selective loss of occludin from tight junctions. It is noteworthy that BBB integrity is reestablished before astrocytes repopulate. Rather than persistent astrocyte loss, polymorphonuclear leukocytes (PMNs) are the main mediators of BBB disruption, and their depletion preserves BBB integrity and prevents astrocyte loss. Inhibition of PMN chemoattraction, activation, and proteolytic function reduces lesion size. In summary, our data support a crucial role for PMNs in BBB disruption and NMOSD lesion development, rendering their recruitment and activation promising therapeutic targets.

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.

Strategy for Treatment of Infected Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are chronic wounds that develop in 30% of diabetic patients. In DFUs, the normal wound healing process consisting of inflammation, angiogenesis, and extracellular matrix (ECM) remodeling is dysregulated and stalled. Upon injury, neutrophils and monocytes arrive at the wound and secrete matrix metalloproteinase (MMP)-8 and reactive oxygen species (ROS). ROS activates nuclear factor kappa beta (NF-κB), which upregulates MMP-9. Monocytes become macrophages, secreting tumor growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF) for angiogenesis, resulting in remodeling of the ECM. MMP-9 cleaves laminin for keratinocyte migration. MMP-8 is beneficial for remodeling the ECM and healing the wound. In DFUs, the excess unregulated MMP-9 is detrimental, destroying the ECM and preventing the wound from healing. DFUs are typically infected, many with biofilm-producing bacteria that are resistant to antibiotics. Infection increases the time for wound healing and the likelihood for a lower-limb amputation. Despite the use of antibiotics, amputations occur in 24.5% of patients with DFUs. Clearly, new strategies for treatment of DFUs are needed. With the use of an affinity resin that binds exclusively to the active forms of MMPs and proteomics, we identified two proteinases, MMP-8 and MMP-9, in wounds of diabetic mice and diabetic humans. With the use of selective inhibitors, gene ablation of MMP-9, and exogenous application of MMP-8, we demonstrated that MMP-8 is beneficial to wound repair and that MMP-9 prevents the diabetic wound from healing. Our research has shown that infection increases active MMP-9, increasing inflammation and decreasing angiogenesis. As a result, infected diabetic wounds take a longer time to heal than uninfected ones. We found that active MMP-9 and NF-κB increased in human DFUs with wound severity and infection. The best strategy for treatment of DFUs is to selectively inhibit the detrimental proteinase MMP-9 without affecting the beneficial MMP-8 so that the body can repair the wound. Lead optimization of the thiirane class of inhibitors led to the discovery of (R)-ND-336, a potent (19 nM) and selective (450-fold) MMP-9 inhibitor. (R)-ND-336 accelerated wound healing in diabetic mice by decreasing ROS and NF-κB, lowering inflammation, and increasing angiogenesis. (R)-ND-336 in combination with the antibiotic linezolid improved wound healing in infected diabetic mice by inhibiting MMP-9, which mitigated macrophage infiltration and increased angiogenesis, thereby restoring the normal wound healing process.

Unconventional Antibacterials and Adjuvants.

The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-ß-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of ß-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a ß-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of ß-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.

Discovery of a Potent Picolinamide Antibacterial Active against Clostridioides difficile.

A major challenge for chemotherapy of bacterial infections is perturbation of the intestinal microbiota. Clostridioides difficile is a Gram-positive bacterium of the gut that can thrive under this circumstance. Its production of dormant and antibiotic-impervious spores results in chronic disruption of normal gut flora and debilitating diarrhea and intestinal infection. C. difficile is responsible for 12,800 deaths per year in the United States. Here, we report the discovery of 2-(4-(3-(trifluoromethoxy)phenoxy)picolinamido)benzo[d]oxazole-5-carboxylate as an antibacterial with potent and selective activity against C. difficile. Its MIC50 and MIC90 (the concentration required to inhibit the growth of 50% and 90% of all the tested strains, respectively) values, documented across 101 strains of C. difficile, are 0.12 and 0.25 µg/mL, respectively. The compound targets cell wall biosynthesis, as assessed by macromolecular biosynthesis assays and by scanning electron microscopy. Animals infected with a lethal dose of C. difficile and treated with compound 1 had a similar survival compared to treatment with vancomycin, which is the frontline antibiotic used for C. difficile infection.

Targeting Extracellular Matrix Remodeling Restores BRAF Inhibitor Sensitivity in BRAFi-resistant Melanoma.

PURPOSE: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy. EXPERIMENTAL DESIGN: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi. RESULTS: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinß1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFß dependent, as inhibition of TGFß receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition. CONCLUSIONS: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.