In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regu lated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM170 s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modula tors as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibi tion mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics

Discovery of Novel Targets for Melanoma Drug Discovery

Objective. To identify target(s) and mechanism of action of novel anti-melanoma lead 2155-14. Background. Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. Methods. We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Results. We identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 2155-14. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. Conclusion. Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets. Grants. Auburn University Harrison School of Pharmacy Faculty start-up funds. TPIMS Faculty start-up funds. Auburn University Intramural Grant Program from Office of Vice President for Research. “Target Identification of Novel Anti-Melanoma Compounds”. Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

Spliceosomal Proteins as Targets for Melanoma Therapy

Objective. To identify target(s) and mechanism of action of novel anti-melanoma lead 2155-14. Background. Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. Methods. We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Clinical significance was analyzed using databases. Results. We identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 2155-14. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. Database analysis showed potential correlation with overall survival of metastatic melanoma patients. Conclusion. Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets. Grants. Auburn University Faculty start-up funds. TPIMS Faculty start-up funds. Auburn University Intramural Grant Program: “Target Identification of Novel Anti-Melanoma Compounds”

In Vivo Efficacy Evaluation of Novel Melanoma Actives

As estimated by the National Cancer Institute (NIH/NCI), there are more than 900,000 people living with melanoma in the USA. Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is ~3 years. Instances of complete response are very rare, therefore, more life-prolonging therapies are needed. This suggests a need for new approaches and targets for melanoma drug discovery. We discovered a novel class of anti-melanoma compounds that potentiate autophagy leading to cell death. Preliminary results in different 2D and 3D cultures of melanoma cells, including BRAF- and NRAS-mutants, demonstrated the efficacy of our leads 2155-14 and 2155-18 comparable to the FDA-approved melanoma drug vemurafenib and the lack of toxicity to non-melanoma cells and a mouse model. Target identification studies revealed that 2155-14 binds to three proteins (DDX1, hnRNPA2/B1, and hnRNPH2) that were not previously considered for melanoma drug discovery. Mechanism of action studies suggest that binding of probes specifically to hnRNPH2 leads to an autophagic cell death in melanoma cells with different mutational backgrounds. This suggests an exciting potential for novel target(s) for broad-spectrum melanoma therapy. The overall aim of this project is to evaluate efficacy of novel anti-melanoma compounds in BRAF and NRAS mutant animal models of melanoma. Our team is uniquely positioned to achieve these goals due to expertise in cancer biochemistry and drug/probe discovery, and animal models. Our expected long-term outcome is to develop safe therapy for melanoma

Production of soluble Neprilysin by endothelial cells

A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 mu M) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 +/- 0.70 and 6.54 +/- 0.41 mu mols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32 mu M, respectively. Treatment of cells with TPI2155-14 (15 mu M) and TPI2155-17 (4.3 mu M) resulted in a significant decrease in NEP activity in media (62.37 +/- 1.43 and 38.30 +/- 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 degrees C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17. (C) 2014 Elsevier Inc. All rights reserved

In Vivo Acute Toxicity Studies of Novel Anti-Melanoma Compounds Downregulators of hnRNPH1/H2

Despite the recent advances in melanoma therapy, the need for new targets and novel approaches to therapy is urgent. We previously reported melanoma actives that work via binding and downregulating spliceosomal proteins hnRNPH1 and H2. Given the lack of knowledge about the side effects of using spliceosomal binders in humans, an acute toxicity study was conducted to evaluate these compounds in mice. Male and female mice were treated with compounds 2155-14 and 2155-18 at 50 mg/kg/day via subcutaneous injections, and the clinical signs of distress were monitored for 21 days and compared with control mice. Additionally, the effect of the leads on blood chemistry, blood cell counts, and organs was evaluated. No significant changes were observed in the body weight, blood cell count, blood chemistry, or organs of the mice following the compound treatment. The results show that our compounds, 2155-14 and 2155-18, are not toxic for the study period of three weeks

Optimization of Immunoprecipitation Protocol of Protein hnRNPH2 from Melanoma Cells

Objective: Despite advances in melanoma drug discovery, the average survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets 1. Introduction: We identified heterogeneous nuclear ribonucleoprotein (hnRNP) H2 as such a target (Palrasu et al., Cell Physiol Biochem 2019;53:656-86). Mechanistic investigations showed that targeting hnRNPH2 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. Our project focused on optimization of isolating and purifying hnRNPH2 from melanoma cells. Methods: Antibody-bead crosslinking, immunoprecipitation (IP), silver stain, Coomassie stain, and western blots were used. Results: Initial experiments showed low levels of isolated H2. In order to obtain greater quantity of the protein, we scaled up antibody/bead complex 10 times resulting in higher H2 levels in eluate. Overall, we were able to isolate 10ug of pure H2. In the future, we are planning to obtain 100ug of pure H2 to perform protein biochemistry and biophysics. Conclusions: It was shown that multiple cycles of IP along with higher scales of antibody/bead complex ratio were required to obtain sufficient amount of the protein to be visible on the western blot stain. 1Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, 3321 College Avenue, CCR r.612, Fort Lauderdale, FL 33314 2 Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 3 Halmos College of Natural Sciences and Oceanography, 8000 N Ocean Dr, Dania Beach, FL 33004 4 Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 1. Foundation MR. Melanoma Facts and Stats 2018. Available from: https://www.melanoma.org/understand-melanoma/what-melanoma/melanoma-facts-and-stats

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (+/- 0.3) x 10(4) M-1 s(-1) and 2.4 (+/- 0.3) x 10(3) M-1 s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAMS and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities. (C) 2016 Elsevier Inc. All rights reserved