Valerie A. Schroeder

ADP-Ribosylation factor 6 regulates tumorigenic and invasive properties in vivo

This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.

Selective Inhibition of Matrix Metalloproteinase-9 Attenuates Secondary Damage Resulting from Severe Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and long-term disability. Following the initial insult, severe TBI progresses to a secondary injury phase associated with biochemical and cellular changes. The secondary injury is thought to be responsible for the development of many of the neurological deficits observed after TBI and also provides a window of opportunity for therapeutic intervention. Matrix metalloproteinase-9 (MMP-9 or gelatinase B) expression is elevated in neurological diseases and its activation is an important factor in detrimental outcomes including excitotoxicity, mitochondrial dysfunction and apoptosis, and increases in inflammatory responses and astrogliosis. In this study, we used an experimental mouse model of TBI to examine the role of MMP-9 and the therapeutic potential of SB-3CT, a mechanism-based gelatinase selective inhibitor, in ameliorating the secondary injury. We observed that activation of MMP-9 occurred within one day following TBI, and remained elevated for 7 days after the initial insult. SB-3CT effectively attenuated MMP-9 activity, reduced brain lesion volumes and prevented neuronal loss and dendritic degeneration. Pharmacokinetic studies revealed that SB-3CT and its active metabolite, p-OH SB-3CT, were rapidly absorbed and distributed to the brain. Moreover, SB-3CT treatment mitigated microglial activation and astrogliosis after TBI. Importantly, SB-3CT treatment improved long-term neurobehavioral outcomes, including sensorimotor function, and hippocampus-associated spatial learning and memory. These results demonstrate that MMP-9 is a key target for therapy to attenuate secondary injury cascades and that this class of mechanism-based gelatinase inhibitor–with such desirable pharmacokinetic properties–holds considerable promise as a potential pharmacological treatment of TBI.

Discovery of a New Class of Non-β-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-β-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.

Synergistic, collaterally sensitive [beta]-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 β-lactam combination meropenem/piperacillin/tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA N315 and 72 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/β-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to β-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein 2a (PBP2a) action via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing similar in vivo activity to linezolid, ME/PI/TZ demonstrates that combinations of older β-lactam antibiotics could be effective against MRSA infections in humans.

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.

Selective Water-Soluble Gelatinase Inhibitor Prodrugs

SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.

Structure−Activity Relationship for the Oxadiazole Class of Antibiotics

The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.

Liposomal Bortezomib Nanoparticles via Boronic Ester Prodrug Formulation for Improved Therapeutic Efficacy in Vivo

In this study, we describe the development of liposomal bortezomib nanoparticles, which was accomplished by synthesizing bortezomib prodrugs with reversible boronic ester bonds and then incorporating the resulting prodrugs into the nanoparticles via surface conjugation. Initially, several prodrug candidates were screened based upon boronic ester stability using isobutylboronic acid as a model boronic acid compound. The two most stable candidates were then selected to create surface conjugated bortezomib prodrugs on the liposomes. Our strategy yielded stable liposomal bortezomib nanoparticles with a narrow size range of 100 nm and with high reproducibility. These liposomal bortezomib nanoparticles demonstrated significant proteasome inhibition and cytotoxicity against multiple myeloma cell lines in vitro and remarkable tumor growth inhibition with reduced systemic toxicity compared to free bortezomib in vivo. Taken together, this study demonstrates the incorporation of bortezomib into liposomal nanoparticles via reversible boronic ester bond formation to enhance the therapeutic index for improved patient outcome.

Liposomal carfilzomib nanoparticles effectively target multiple myeloma cells and demonstrate enhanced efficacy in vivo

Carfilzomib, a recently FDA-approved proteasome inhibitor, has remarkable anti-myeloma (MM) activity. However, its effectiveness is limited by associated severe side-effects, short circulation half-life, and limited solubility. Here, we report the engineering of liposomal carfilzomib nanoparticles to overcome these problems and enhance the therapeutic efficacy of carfilzomib by increasing tumoral drug accumulation while decreasing systemic toxicity. In our design, carfilzomib was loaded into the bilayer of liposomes to yield stable and reproducible liposomal nanoparticles. Liposomal carfilzomib nanoparticles were efficiently taken up by MM cells, demonstrated proteasome inhibition, induced apoptosis, and exhibited enhanced cytotoxicity against MM cells. In vivo, liposomal carfilzomib demonstrated significant tumor growth inhibition and dramatically reduced overall systemic toxicity compared to free carfilzomib. Finally, liposomal carfilzomib demonstrated enhanced synergy in combination with doxorubicin. Taken together, this study establishes the successful synthesis of liposomal carfilzomib nanoparticles that demonstrates improved therapeutic index and the potential to improve patient outcome in MM.

Structure-Activity Relationship for the 4(3H)-Quinazolinone Antibacterials.

We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity against Staphylococcus aureus. The first structure-activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.