Stimulation of natural killer cells with small molecule inhibitors of CD38 for the treatment of neuroblastoma.

High-risk neuroblastoma (NB) accounts for 15% of all pediatric cancer deaths. Refractory disease for high-risk NB patients is attributed to chemotherapy resistance and immunotherapy failure. The poor prognosis for high-risk NB patients demonstrates an unmet medical need for the development of new, more efficacious therapeutics. CD38 is an immunomodulating protein that is expressed constitutively on natural killer (NK) cells and other immune cells in the tumor microenvironment (TME). Furthermore, CD38 over expression is implicated in propagating an immunosuppressive milieu within the TME. Through virtual and physical screening, we have identified drug-like small molecule inhibitors of CD38 with low micromolar IC50 values. We have begun to explore structure activity relationships for CD38 inhibition through derivatization of our most effective hit molecule to develop a new compound with lead-like physicochemical properties and improved potency. We have demonstrated that our derivatized inhibitor, compound 2, elicits immunomodulatory effects in NK cells by increasing cell viability by 190 ± 36% in multiple donors and by significantly increasing interferon gamma. Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. Herein we describe the synthesis and biological evaluation of small molecule CD38 inhibitors and demonstrate their potential utility as a novel approach to NB immunotherapy. These compounds represent the first examples of small molecules that stimulate immune function for the treatment of cancer.

Selective targeting of CD38 hydrolase and cyclase activity as an approach to immunostimulation.

The ectoenzyme CD38 is highly expressed on the surface of mature immune cells, where they are a marker for cell activation, and also on the surface of multiple tumor cells such as multiple myeloma (MM). CD38-targeted monoclonal antibodies (MABs) such as daratumumab and isatuximab bind to CD38 and promote cancer cell death by stimulating the antitumor immune response. Although MABs are achieving unprecedented success in a percentage of cases, high rates of resistance limit their efficacy. Formation of the immunosuppressive intermediate adenosine is a major route by which this resistance is mediated. Thus there is an urgent need for small molecule agents that boost the immune response in T-cells. Importantly, CD38 is a dual-function enzyme, serving as a hydrolase and a nicotinamide adenine dinucleotide (NAD+) cyclase, and both of these activities promote immunosuppression. We have employed virtual and physical screening to identify novel compounds that are selective for either the hydrolase or the cyclase activity of CD38, and have demonstrated that these compounds activate T cells in vitro. We are currently optimizing these inhibitors for use in immunotherapy. These small molecule inhibitors of the CD38-hydrolase or cyclase activity can serve as chemical probes to determine the mechanism by which CD38 promotes resistance to MAB therapy, and could become novel and effective therapeutic agents that produce immunostimulatory effects. Our studies have identified the first small molecule inhibitors of CD38 specifically for use as immunostimulants.

A fluorescent approach for detecting and measuring reduction reaction byproducts near cathodically-biased metallic surfaces: Reactive oxygen species production and quantification.

During tribocorrosion of biomedical alloys, potentials may shift cathodically across the metal-oxide-electrolyte interface resulting in the increased reduction of local oxygen and water molecules. The products of reduction are thought to include reactive oxygen species (ROS) as well as hydroxide ions. Using fluorescent probes, developed for labeling intracellular ROS-based hydroxyl radicals (OH·) and hydrogen peroxide (H2O2), ROS generation due to reduction reactions at cathodically biased CoCrMo alloy surfaces was measured directly. Using terephthalic acid (TA) and pentafluorosulfonylbenzene-fluorescein (PFF) as fluorescent dosimeters, it was found that OH· and H2O2 concentrations increased up to 16 h and 2 h, respectively. Decreases in fluorescence past these time points were attributed to the continuous onset of reduction reactions consuming both the ROS and/or dosimeter. It was also found that voltages below and including -600 mV (vs. Ag/AgCl) produced measurable quantities of H2O2 after two hours of polarization, with concentrations increasing with decreasing potentials up to -1000 mV. The detection and quantification of ROS in a clinical setting could help us better understand the role of ROS in the inflammatory response as well as their impact on corrosion behavior of biomedical alloys.

Microbiota-Dependent Metabolite Trimethylamine N-Oxide and Coronary Artery Calcium in the Coronary Artery Risk Development in Young Adults Study (CARDIA).

BACKGROUND: Clinical studies implicate trimethylamine N-oxide (TMAO; a gut microbiota-dependent nutrient metabolite) in cardiovascular disease risk. There is a lack of population-based data on the role of TMAO in advancing early atherosclerotic disease. We tested the prospective associations between TMAO and coronary artery calcium (CAC) and carotid intima-media thickness (cIMT). METHODS AND RESULTS: Data were from the Coronary Artery Risk Development in Young Adults Study (CARDIA), a biracial cohort of US adults recruited in 1985-1986 (n=5115). We randomly sampled 817 participants (aged 33-55 years) who attended examinations in 2000-2001, 2005-2006, and 2010-2011, at which CAC was measured by computed tomography and cIMT (2005-2006) by ultrasound. TMAO was quantified using liquid chromotography mass spectrometry on plasma collected in 2000-2001. Outcomes were incident CAC, defined as Agatston units=0 in 2000-2001 and >0 over 10-year follow-up, CAC progression (any increase over 10-year follow-up), and continuous cIMT. Over the study period, 25% (n=184) of those free of CAC in 2000-2001 (n=746) developed detectable CAC. In 2000-2001, median (interquartile range) TMAO was 2.6 (1.8-4.2) µmol/L. In multivariable-adjusted models, TMAO was not associated with 10-year CAC incidence (rate ratio=1.03; 95% CI: 0.71-1.52) or CAC progression (0.97; 0.68-1.38) in Poisson regression, or cIMT (beta coefficient: -0.009; -0.03 to 0.01) in linear regression, comparing the fourth to the first quartiles of TMAO. CONCLUSIONS: In this population-based study, TMAO was not associated with measures of atherosclerosis: CAC incidence, CAC progression, or cIMT. These data indicate that TMAO may not contribute significantly to advancing early atherosclerotic disease risk among healthy early-middle-aged adults.