Evaluating the Toxicity of the Analgesic Glutaminase Inhibitor 6-Diazo-5-Oxo-L-Norleucine in vitro and on Rat Dermal Skin Fibroblasts

Abstract

6-diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist produced naturally by Streptomyces. It inhibits several glutamine-dependent enzyme pathways. Of particular note is its inhibitory effect on the mitochondrial enzyme, glutaminase (GLS), the primary producer of neuronal glutamate. Glutamate is an excitatory neurotransmitter released by primary sensory peripheral nerve terminals and spinal synaptic terminals during pain signaling. Previous work using the tail incision and inflammatory models of pain has demonstrated that a single application of the glutaminase inhibitor, DON, into a surgical incision or the paw of arthritic animals results in pain relief. Even though this compound shows promise as a therapeutic agent, limited data exist regarding its dermal toxicity. As a first approach, we evaluated the effect of several concentrations of DON, on the viability, mitochondrial oxidative capacity and proliferation of rat skin fibroblasts, and then examined the effect of DON after incubation with human liver microsomes on proliferation. Finally, we evaluated DON treated rat skin (tail and hind paw) for cellular necrosis, inflammation and mitotic bodies. No significant effects (p > 0.05) of DON were noted on apoptosis, necrosis, and mitochondrial activity in experiments with cultured rat skin fibroblasts. Flow cytometry revealed the absence of apoptosis in cells treated at the IC50 of 232.5 μM. Enhanced toxicity post-exposure to human microsomes was not observed when compared to DON alone. The H&E staining of the rat skin revealed no obvious pathology in the DON treatment group (10 mM). DON has no/minimal cellular toxicity in vitro on dermal fibroblasts at concentrations that effectively provide analgesia. The local application of concentrations greater than the in vitro IC50 for DON revealed no in vivo skin toxicity. These data provide results indicating zero-to-minimal cellular toxicity with DON and support the further investigation of DON as an analgesic.

Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach

“…Several derivations or analogs of BPTES [ 112 ] and a newly discovered glutaminase 1 inhibitor CB-839 are also being researched. The glutamine antagonist such as DON (6-diazo-5-oxo-l-norleucine) found previously is nonspecific, has toxic or side effects, and inhibits several enzymes of glutamine utilization [ 96 ]. Drugs such as ebselen, chelerythrine and apomorphine were also regarded as GLS inhibitors in a recent research ( Table 1 ) [ 95 ].…”

Dysregulation of Astrocytic Glutamine Transport in Acute Hyperammonemic Brain Edema

“…Upon entry to mitochondria, glutamine is metabolized by the mitochondrial enzyme PAG to ammonia and glutamate and, ammonia is the trigger of subsequent deleterious events. This sequence of events has been documented in cultured astrocytes, where the glutamine-induced intra-astrocytic accumulation of reactive oxygen species ( Jayakumar et al, 2004 ) and astrocytic swelling ( Jayakumar et al, 2006 ) were attenuated, by the PAG synthetic inhibitor, DON ( Crosby, 2015 ).…”

Nerve Growth Factor Signaling Modulates the Expression of Glutaminase in Dorsal Root Ganglion Neurons during Peripheral Inflammation

“…The inhibitors were injected and topically applied once before 30 mins of CFA treatment and two times: 12 hours and 36 hours after CFA treatment. Rats with respective volume PBS injections as compared to treated were used as controls [52].…”