Activated RAS is a common driver of cancer that was considered undruggable for decades. Recent advances have enabled the development of RAS inhibitors, but the efficacy of these inhibitors remains limited by resistance. In this study, we developed a pan-RAS inhibitor, ADT-007, (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide, that binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 potently inhibited the growth of RAS-mutant cancer cells irrespective of the RAS mutation or isozyme. Wild-type RAS (RASWT) cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, whereas insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases that were expressed in RASWT and normal cells but repressed in RAS-mutant cancer cells. ADT-007 displayed unique advantages over KRAS mutant-specific, pan-KRAS, and pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms that lead to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immunocompetent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancers. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth. Thus, ADT-007 has the potential to address the complex RAS mutational landscape of many human cancers and to improve treatment of RAS-driven tumors.Significance: ADT-007, a first-in-class pan-RAS inhibitor, has unique selectivity for cancer cells with mutant RAS or activated RAS protein and the capability to circumvent resistance to suppress tumor growth, supporting further development of ADT-007 analogs.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RAS WT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases expressed in RAS WT and normal cells but repressed in RAS mutant cancer cells. ADT-007 binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 displayed unique advantages over mutant-specific KRAS and pan-KRAS inhibitors, as well as other pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms leading to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for RAS-driven cancers. SIGNIFICANCE:ADT-007 has unique pharmacological properties with distinct advantages over other RAS inhibitors by circumventing resistance and activating antitumor immunity. ADT-007 prodrugs and analogs with oral bioavailability warrant further development for RAS-driven cancers.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, Fe-57-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), Fe-57 electron-nuclear double resonance (ENDOR), and M & ouml;ssbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the Fe-57 hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S](2+) cluster. Reduction generates [4Fe-4S](+) (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the M & ouml;ssbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S](+) species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while M & ouml;ssbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

ABSTRACT Here we describe a novel class of pan-RAS inhibitor with highly potent and selective anticancer activity by killing cancer cells harboring mutations in RAS or with constitutively activated RAS resulting from mutations in upstream signaling components. A lead compound from this chemical family, ADT-007, binds RAS when in a nucleotide free transitional state to block loading of GTP, thereby interfering with RAS activation and disruption of binding to effectors such as RAF and PI3K to suppress MAPK and AKT signaling. ADT-007 potently inhibits the growth of cultured human and murine cancer cell lines with single-digit nM IC50 values irrespective of specific RAS isozyme or mutational codon. ADT-007 also inhibits tumor growth in vivo through inhibition of RAS-MAPK signaling in syngeneic, immune competent and xenogeneic, immune deficient mouse models of colon and pancreatic cancer. In RAG 1 -/- mice the activity of ADT-007 is partially inhibited indicating a role for the adaptive immune system in ADT-007-mediated tumor growth inhibition. Ex vivo analyses of tumor infiltrating leukocytes, reveals that ADT-007 enhances T cell functions in the pancreatic and colorectal tumor immune microenvironment. SIGNIFICANCE ADT-007 represents a 1st-in-class pan-RAS inhibitor with broad anticancer activity across all RAS driven cancers and unique chemical selectivity to allow normal cells to be spared from pan-RAS inhibition. ADT-007 also has the potential to modulate the adaptive immune response in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA). These data support future clinical trials of an orally bioavailable prodrug of ADT-007 as a monotherapy for the treatment of patients with CRC or PDAC regardless of the underlying mutation or in combination with immunotherapy.

RAS is a critically important oncogenic protein that is mutated in approximately 1/3 of cancers resulting in aberrant activation of downstream signaling, which drives malignant transformation. Current molecular targeted therapeutics, and several in development, inhibit only specific mutant alleles (G12C, G12D). In addition, compounds which directly inhibit RAS via proteolytic degradation or inhibit RAS activation by SOS1 are in preclinical development and are referred to as Pan-KRAS inhibitors. Previously investigators have reported that the NSAID, sulindac, can selectively inhibit RAS mutant tumorigenesis by a cyclooxygenase (COX)-independent mechanism. Sulindac, and more potent analogs have also previously been reported to inhibit RAS-mediated transformation and directly bind RAS. Here we describe an ultra-potent non-COX inhibitory derivative of sulindac, ADT-007, which binds to and inhibits RAS nucleotide binding and RAS-effector association. ADT-007 binding to KRAS was evaluated by Micro-Tag cell target engagement and by Cellular Thermal Shift Assay (CETSA) which demonstrated a potency of target engagement (EC50) value in the subnanomolar range. Consistent with molecular docking studies, HSQC NMR spectroscopy using recombinant KRAS revealed that ADT-007 interacted with KRAS after Mg2+ chelation to obtain a nucleotide free (NF) state, resulting in chemical shift changes and signal attenuation of residues in the P-loop and nucleotide binding domain. Similarly, biochemical assays confirmed that ADT-007 prevented MANT-GTP binding to recombinant NF KRAS but did not compete with bound GTP. Functional assays also showed that KRAS binding to RAF-RBD(GST) was inhibited by ADT-007. The compound inhibited constitutive RAS activation (RAF-RBD pulldown) in serum starved MiaPaCa-2 pancreatic cancer cells harboring a KRAS-G12C mutation and demonstrated Pan-RAS inhibition in serum- or EGF-stimulated cells. Further, ADT-007 inhibited AKT phosphorylation and EGF-stimulated downstream ERK1/2 phosphorylation. Finally, ADT-007 demonstrated RAS-selective growth inhibition in isogenic pancreatic and colorectal cancer cell pairs (BxPC-3, HT29). Together, these experiments support further development of ADT-007 and related analogs for treatment of RAS-driven cancers. Citation Format: Adam B. Keeton, Xi Chen, Jacob Valiyaveettil, Chung-Hui Huang, Tyler E. Mattox, Khalda Fadlalla, Jeremy B. Foote, Donald J. Buchsbaum, Kristy L. Berry, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Gregory Gorman, Lori Coward, Yulia Y. Maxuitenko, Forrest T. Smith, Gary A. Piazza. ADT-007 binds RAS and inhibits RAS signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1658.

Abstract RAS is a critically important oncogenic protein that is mutated in approximately 1/3 of cancers resulting in aberrant activation of downstream signaling, which drives malignant transformation. Current molecular targeted therapeutics, and several in development, inhibit only specific mutant alleles (G12C, G12D). In addition, compounds which directly inhibit RAS via proteolytic degradation or inhibit RAS activation by SOS1 are in preclinical development and are referred to as Pan-KRAS inhibitors. Previously investigators have reported that the NSAID, sulindac, can selectively inhibit RAS mutant tumorigenesis by a cyclooxygenase (COX)-independent mechanism. Sulindac, and more potent analogs have also previously been reported to inhibit RAS-mediated transformation and directly bind RAS. Here we describe an ultra-potent non-COX inhibitory derivative of sulindac, ADT-007, which binds to and inhibits RAS nucleotide binding and RAS-effector association. ADT-007 binding to KRAS was evaluated by Micro-Tag cell target engagement and by Cellular Thermal Shift Assay (CETSA) which demonstrated a potency of target engagement (EC50) value in the subnanomolar range. Consistent with molecular docking studies, HSQC NMR spectroscopy using recombinant KRAS revealed that ADT-007 interacted with KRAS after Mg2+ chelation to obtain a nucleotide free (NF) state, resulting in chemical shift changes and signal attenuation of residues in the P-loop and nucleotide binding domain. Similarly, biochemical assays confirmed that ADT-007 prevented MANT-GTP binding to recombinant NF KRAS but did not compete with bound GTP. Functional assays also showed that KRAS binding to RAF-RBD(GST) was inhibited by ADT-007. The compound inhibited constitutive RAS activation (RAF-RBD pulldown) in serum starved MiaPaCa-2 pancreatic cancer cells harboring a KRAS-G12C mutation and demonstrated Pan-RAS inhibition in serum- or EGF-stimulated cells. Further, ADT-007 inhibited AKT phosphorylation and EGF-stimulated downstream ERK1/2 phosphorylation. Finally, ADT-007 demonstrated RAS-selective growth inhibition in isogenic pancreatic and colorectal cancer cell pairs (BxPC-3, HT29). Together, these experiments support further development of ADT-007 and related analogs for treatment of RAS-driven cancers. Citation Format: Adam B. Keeton, Xi Chen, Jacob Valiyaveettil, Chung-Hui Huang, Tyler E. Mattox, Khalda Fadlalla, Jeremy B. Foote, Donald J. Buchsbaum, Kristy L. Berry, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Gregory Gorman, Lori Coward, Yulia Y. Maxuitenko, Forrest T. Smith, Gary A. Piazza. ADT-007 binds RAS and inhibits RAS signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1658.

Purpose: Difluprednate (DFP) is an approved corticosteroid, available as an ophthalmic emulsion (Durezol®), used to treat pain and inflammation of the eye following ocular surgeries. This study utilized hydroxypropyl-β-cyclodextrin (HPBCD)-based DFP ophthalmic solution for improved ocular delivery. Methods: The DFP-HPBCD complex formation was studied in the liquid and solid states. Phase solubility, molecular docking studies, differential scanning calorimetry, and Fourier transform infrared spectroscopy suggested inclusion complexation of DFP and HPBCD. Results: DFP-HPBCD-based eye drops (solution) provided 16 and 26 times higher transcorneal permeation when compared to the suspension (no HPBCD, control) and Durezol, respectively (P < 0.001). In addition, ocular drug distribution studies conducted in continuously perfused whole porcine eyes showed DFP permeated into all of the ocular tissues in significantly higher amounts than Durezol. Conclusions: The solution-based eye drops in this study is iso-osmotic, safe, and more permeable in porcine eyes compared to Durezol.

Background and purpose: Medication-induced cardiotoxicity is a significant factor in the attrition of drugs during preclinical and clinical development processes. Patients with diabetes mellitus (hyperglycemic) are more than twice as likely to experience cardiac failure. Additionally, type 2 diabetes mellitus (T2D) patients often display significant hyperarousal-related clinical anomalies such as fear, panic, nervousness, pain, and seizures. Consequently, hyperarousal in patients with inadequate metabolic outcomes (hyperglycemic conditions) is usually treated with drugs that block sodium/calcium channels, augment inhibitory (gamma-aminobutyric acid [GABA]) neurotransmission, and reduce excitatory (glutamatergic) neurotransmission. These perilous combined clinical-pathological conditions of hyperglycemia and hypoarousal may result in severe learning disabilities and cognitive impairment. Unfortunately, only a few studies have investigated the synergistic effects of hypoarousal and hyperglycemia on cognition. Methods: General behavioral assessment, plus maze, Y-maze spontaneous alternation, Hebb-Williams maze and Passive avoidance paradigm were evaluated in this study. The current study assessed the in silico structural properties attributed to its pharmacodynamic actions and interaction with Gamma-aminobutyric acid (GABA) and insulin receptors using Schrodinger and LigPrep software. Results: The administration of alloxan and phenytoin induced significant learning and cognitive deficiencies. Based on the in silico studies, alloxan is a better drug to induce hyperglycemia as compared to the well-established hyperglycemic agent, streptozotocin (STZ). Conclusions: The current study indicated that administering alloxan and phenytoin to rodents can serve as a valid animal model to understand the pathophysiology associated with hypoarousal and hyperglycemia-mediated cognitive impairment and to identify novel therapeutic interventions for hyperglycemic and hypoarousal-related learning and cognitive deficiency.

Hispolon, a phenolic pigment isolated from the mushroom species Phellinus linteus, has been investigated for anti-inflammatory, antioxidant, and anticancer properties; however, low solubility and poor bioavailability have limited its potential clinical translation. In this study, the inclusion complex of hispolon with Sulfobutylether-β-cyclodextrin (SBEβCD) was characterized, and the Hispolon-SBEβCD Complex (HSC) was included within the sterically stabilized liposomes (SL) to further investigate its anticancer activity against melanoma cell lines. The HSC-trapped-Liposome (HSC-SL) formulation was investigated for its sustained drug delivery and enhanced cytotoxicity. The inclusion complex in the solid=state was confirmed by a Job’s plot analysis, molecular modeling, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), Proton nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). The HSC-SL showed no appreciable deviation in size (<150 nm) and polydispersity index (<0.2) and improved drug encapsulation efficiency (>90%) as compared to control hispolon liposomes. Individually incorporated hispolon and SBEβCD in the liposomes (H-CD-SL) was not significant in loading the drug in the liposomes, compared to HSC-SL, as a substantial amount of free drug was separated during dialysis. The HSC-SL formulation showed a sustained release compared to hispolon liposomes (H-SLs) and Hispolon-SBEβCD liposomes (H-CD-SLs). The anticancer activity on melanoma cell lines (B16BL6) of HSC and HSC-SL was higher than in H-CD-SL and hispolon solution. These findings suggest that HSC inclusion in the HSC-SL liposomes stands out as a potential formulation approach for enhancing drug loading, encapsulation, and chemotherapeutic efficiency of hispolon and similar water insoluble drug molecules.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and its incidence is increasing worldwide with increased lifespan. Currently, there is no effective treatment to cure or prevent the progression of AD, which indicates the need to develop novel therapeutic targets and agents. Sirtuins, especially SIRT3, a mitochondrial deacetylase, are NAD-dependent histone deacetylases involved in aging and longevity. Accumulating evidence indicates that SIRT3 dysfunction is strongly associated with pathologies of AD, hence, therapeutic modulation of SIRT3 activity may be a novel application to ameliorate the pathologies of AD. Natural products commonly used in traditional medicine have wide utility and appear to have therapeutic benefits for the treatment of neurodegenerative diseases such as AD. The present review summarizes the currently available natural SIRT3 activators and their potentially neuroprotective molecular mechanisms of action that make them a promising agent in the treatment and management of neurodegenerative diseases such as AD.

The widespread increase in multiple severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants is causing a significant health concern in the United States and worldwide. These variants exhibit increased transmissibility, cause more severe disease, exhibit evasive immune properties, impair neutralization by antibodies from vaccinated individuals or convalescence sera, and reinfection. The Centers for Disease Control and Prevention (CDC) has classified SARS-CoV-2 variants into variants of interest, variants of concern, and variants of high consequence. Currently, four variants of concern (B.1.1.7, B.1.351, P.1, and B.1.617.2) and several variants of interests (B.1.526, B.1.525, and P.2) are characterized and are essential for close monitoring. In this review, we discuss the different SARS-CoV-2 variants, emphasizing variants of concern circulating the world and highlight the various mutations and how these mutations affect the characteristics of the virus. In addition, we discuss the most common vaccines and the various studies concerning the efficacy of these vaccines against different variants of concern.

Administration of Chemotherapeutics, especially doxorubicin (DOX) and cyclophosphamide (CPS), is commonly associated with adverse effects such as myelosuppression and cardiotoxicity. At this time, few approved therapeutic options are currently available for the management of chemotherapy-associated cardiotoxicity. Thus, identification of novel therapeutics with potent cardioprotective properties and minimal adverse effects are pertinent in treating Doxorubicin and Cyclophosphamide-induced cardiotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) is a natural product known to possess several beneficial biological functions including antioxidant, anti-inflammatory and cytoprotective effects. We therefore set to investigate the cardioprotective effects of OIE against Doxorubicin and Cyclophosphamide-induced cardiotoxicity and explore the potential cardioprotective mechanisms involved. Adult male mice were treated with DOX and CPS in combination, OIE alone, or a combination of OIE and DOX & CPS. Swimming test was performed to assess cardiac function. Markers of oxidative stress were assessed by levels of reactive oxygen species (ROS), nitrite, hydrogen peroxide, catalase, and glutathione content. The activity of interleukin converting enzyme and cyclooxygenase was determined as markers of inflammation. Mitochondrial function was assessed by measuring Complex-I activity. Apoptosis was assessed by Caspase-3 and protease activity. Mice treated with DOX and CPS exhibited reduced swim rate, increased oxidative stress, increased inflammation, and apoptosis in the heart tissue. These cardiotoxic effects were significantly reduced by co-administration of OIE. Furthermore, computational molecular docking studies revealed potential binding of DOX and CPS to tyrosine hydroxylase which validated our in vivo findings regarding the inhibition of tyrosine hydroxylase activity. Our current findings indicated that OIE counteracts Doxorubicin and Cyclophosphamide-induced cardiotoxicity-through inhibition of ROS-mediated apoptosis and by blocking the effect on tyrosine hydroxylase. Taken together, our findings suggested that OIE possesses cardioprotective effects to counteract potentially fatal cardiac complications associated with chemotherapy treatment.

Ketone bodies have been the topic of research for their possible therapeutic neurotropic effects in various neurological diseases such as Parkinson's disease, dementia, and seizures. However, continuing research on ketone bodies as a prophylactic agent for decreasing the risk for various neurodegenerative diseases is currently required. In this paper, hippocampal HT-22 cells were treated with β-hydroxybutyric acid at different doses to elucidate the neurotropic effects. In addition, markers of oxidative stress, mitochondrial function, and apoptosis were investigated. As a result, the ketone body (β-hydroxybutyric acid) showed a significant increase in hippocampal neuronal viability at a moderate dose. Results show that β-hydroxybutyric acid exhibited antioxidant effect by decreasing prooxidant oxidative stress markers such as reactive oxygen species, nitrite content, and increasing glutathione content leading to decreased lipid peroxidation. Results show that β-hydroxybutyric acid improved mitochondrial functions by increasing Complex-I and Complex-IV activities and showing that β-hydroxybutyric acid significantly reduces caspase-1 and caspase-3 activities. Finally, using computational pharmacokinetics and molecular modeling software, we validated the pharmacokinetic effects and pharmacodynamic (N-Methyl-D-aspartic acid and acetylcholinesterase) interactions of β-hydroxybutyric acid. The computational studies demonstrate that β-hydroxybutyric acid can interact with N-Methyl-D-aspartic acid receptor and cholinesterase enzyme (the prime pharmacodynamic targets for cognitive impairment) and further validates its oral absorption, distribution into the central nervous system. Therefore, this work highlights the neuroprotective potential of ketone bodies in cognitive-related neurodegenerative diseases.

Chronic inflammation is a key culprit factor in the onset and progression of several diseases. Novel and pharmacologically effective therapeutic approaches are needed for new treatment remedy or improved pharmacokinetics and pharmacodynamics for existing synthetic drugs, in particular natural products. Boswellic acids are well-known natural products, with capacity to effectively retard inflammation without severe adverse effects. However, the therapeutic use of Boswellic acids are greatly hindered by its poor pharmacokinetic properties. Co-administration strategies that facilitate the oral absorption and distribution of Boswellic acids should lead to a safe and more effective use of this product prophylactically and therapeutically in inflammatory disorders. In this study, we examined the effect of Piper longum extract on the absorption and bioavailability of Boswellic acid in rabbits. In addition, we further explored computational pharmacodynamic interactions between Piper longum and Boswellic acid. Piper longum extract at 2.5 and 10 mg/kg, increased the bioavailability of Boswellic acid (p < 0.05). Based on our drug-based computational modeling, cytochrome P450 (CYP450)-mediated mechanism was involved in increased bioavailability. These findings confirmed that Piper longum with Boswellic acid may be administered orally together for effective therapeutic efficacy. Thus, our studies support the application of Piper longum with Boswellic acid as a novel therapeutic avenue in diseases associated with inflammation.

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2β) functions as a partial agonist at ErbB4. NRG2β/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

The six 1-n-pentyl-2-, 3-, 4-, 5-, 6- and 7-(2-naphthoyl)-indoles each have the same substituents attached to the indole ring, identical elemental composition (C24H23NO) yielding identical nominal and accurate masses. The electron ionization mass spectra of the 2-naphthoyl substituted isomers share equivalent major fragment ions resulting from cleavage of the groups attached to the central indole nucleus with some differences in relative abundances. These six regioisomers were successfully resolved on an Rtx-5 and Rxi-17Sil MS stationary phases and the molecules having both substituent groups on the same side of the indole ring (1,2- and 1,7-substituents) show the least retention. The more linear molecules have higher relative retention properties. A comparison of the GC properties of the 1-naphthoyl- and 2-naphthoyl groups attached at identical positions of the indole ring showed higher GC retention for the 2-naphthoyl substituted isomer in all cases evaluated. The amide inverse isomers (1-naphthoyl-3-n-pentylindoles) were separated from the 1-n-pentyl-3-naphthoyl-indoles on an Rtx-200 stationary phase. The two inverse amide isomers having the 1- and 2-naphthoyl groups substituted at the 1-position of the indole ring elute before either of the N-alkyl-indole isomers having the 1- and 2-naphthoyl groups substituted at the 3-position of the indole ring. The amide inverse isomers yield EI mass spectra easily distinguishing these amides from the ketone isomers having the naphthoyl groups at the indole 3-position.

Nepafenac is a nonsteroidal anti-inflammatory drug (NSAID), currently only available as 0.1% ophthalmic suspension (Nevanac®). This study utilized hydroxypropyl-β-cyclodextrin (HPBCD) to increase the water solubility and trans-corneal permeation of nepafenac. The nepafenac-HPBCD complexation in the liquid and solid states were confirmed by phase solubility, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR) analyses. Nepafenac 0.1% ophthalmic solution was formulated using HPBCD (same pH and osmolality as that of Nevanac®) and pig eye trans-corneal permeation was studied versus Nevanac®. Furthermore, nepafenac content in cornea, sclera, iris, lens, aqueous humor, choroid, ciliary body, retina, and vitreous humor was studied in a continuous isolated pig eye perfusion model in comparison to the suspension and Nevanac®. Permeation studies using porcine corneas revealed that the solution formulation had a permeation rate 18 times higher than Nevanac®. Furthermore, the solution had 11 times higher corneal retention than Nevanac®. Drug distribution studies using porcine eyes revealed that the solution formulation enables detectable levels in various ocular tissues while the drug was undetectable by Nevanac®. The ocular solution formulation had a significantly higher drug concentration in the cornea compared to the suspension or Nevanac®.

The indole ring regioisomeric methoxy-1-n-pentyl-3-(1-naphthoyl)-indoles represent indole ring-substituted analogs of the synthetic cannabinoid JWH-018. The electron ionization mass spectra show equivalent regioisomeric major fragments resulting from cleavage of the groups attached to the central indole nucleus. The characteristic (M-17)(+) fragment ion at m/z 354 resulting from the loss of OH group is significant in the mass spectra of all four compounds. Fragmentation of the naphthoyl and/or pentyl groups yields the cations at m/z 314, 300, 244 and 216. The vapor-phase infrared spectra provide a number of characteristic absorption bands to identify the individual isomers. Gas chromatographic separations on a capillary column containing a film of trifluoropropylmethyl polysiloxane (Rtx-200) provided excellent resolution of these compounds, their precursor indoles and intermediate pentylindoles. The elution order appears related to the degree of crowding of indole ring substituents.