Microglia play important roles in neutralizing infectious pathogens, responding to traumatic or ischemic damage, and shaping synaptic/dendritic elements under conditions of neuroplasticity. However, excessive activation of microglia under conditions generally characterized as neuroinflammation can contribute to neurodegeneration [21]. Though neuroinflammation is a complex phenomenon associated with release of cytokines and other factors, a potent component of their neurotoxicity is the release of excitatory amino acid transmitters through SxC− [2, 11, 22, 23]. Therefore, we have pursued the development of chemical agents that can inhibit SxC− transport, anticipating neuroprotection during conditions of neuroinflammation. Approximately, a dozen compounds were designed, synthesized, and tested. LPS was used to activate microglia and induce their release of glutamate and uptake of cystine via SxC−. While microglia have been reported to release glutamate via connexin hemichannels [18], this mechanism does not appear to contribute to the glutamate release under the conditions reported here, which is sensitive to α-aminoadipic acid, lipophilic antioxidants, protein kinase A inhibition, and removal of cystine from the medium [5, 14]. Moreover, two members of the novel compounds were confirmed to inhibit cystine uptake. The analysis of 35DBTA7 and 35DBTSA12 was later extended to assays of neuroprotection in the face of activated microglia. Both compounds displayed their ability to reduce microglial release of excitotoxic levels of Glu in vitro.

Our early structure–activity relationship (SAR) studies implied that there is a large lipophilic pocket in the SxC− protein on the side of the α-amino substituent of the inhibitors. After identification of the first inhibitor lead (3,5-DBT), a panel of new molecules for SAR studies was designed and prepared. Remarkable activity of 35DBTA3 (as compared to 35DBTA1, 35DBTA2, and 35DBTA4) drew the attention to the importance of the lipophilic substituent in the para-position of the aromatic system of the amide part. That position was later thoroughly explored. The new molecules, designated 35DBTA7 and 35DBTA8 were quite effective at inhibiting microglial glutamate release. It is important to point out that, besides the pharmacokinetics, one of the key factors guiding us in selection of promising inhibitors is their action at submicromolar concentrations. One inhibitor that did not follow trends was 35DBTA6, a trifluoroacetamide of 3,5-DBT. This molecule seemed to trend toward inhibitory properties at low concentrations but produced a reversal of that trend above 3 μM. Other agents that cause cell death elevate extracellular Glu, presumably due to escape from the cytosol of lysed cells. Thus, the biphasic nature of 35DBTA6 may reflect toxicity at higher concentrations, a common finding with fluorine derivatives. Because one of the concerns was the possibility of premature hydrolysis of the new inhibitors (the amide bond), a stable sulfonamide 35DBTSA12 (a direct sulfo analog of 35DBTA3) was designed and tested. Even though its activity was similar to that of 35DBTA7, it showed diminished potency in the neuroprotection assay. 3,5-DIT displayed promising potency but proved to be exceedingly light sensitive and, therefore, less appealing.

To extend the relevance of the in vitro screening, the two compounds were tested in an assay of neuroprotection. Although other factors have been identified that mediate neurotoxicity in neuroinflammation, we find that Glu receptor agonists are among the most potent. For instance, neuroprotection in a microglia–neuron coculture was far superior with an inhibitor of nitric oxide synthase (NOS) 1—the neuronal isoform activated by Glu receptor agonism—than with an inhibitor of NOS2—the isoform expressed in activated microglia [4]. It is possible that Glu released via SxC− is potentiated by other microglia-derived Glu receptor ligands, such as D-serine or quinolinic acid [24, 25]. In addition, cytokines, reactive oxygen species, and proteases may play roles over distances in space and time [21]. Nevertheless, we and others find that a substantial fraction of microglial neurotoxicity can be alleviated by blocking the effects of Glu. Here, we found that 35DBTA7 was quite effective in such a model of indirect neurotoxicity. The lower efficacy of 35DBTSA12 may indicate that it exerts a mild degree of direct neurotoxicity of its own. Alternatively, it is possible that 35DBTA7 actually served as a prodrug, hydrolyzing to 3,5-DBT for greatest efficacy. If so, this relationship might be exploited in vivo, for instance, to delay the formation of 3,5-DBT until the prodrug has crossed the blood–brain barrier, thus trapping and concentrating the effective agent in the CNS.

Because of their remarkable potency, 35DBTA3 and 35DBTA8 seem worth investigating further. However, 35DBTA8, being the best inhibitor from an activity standpoint, is computationally classified as having poor bioavailability, possibly not crossing the GI tract membrane (Fig. 4). In addition, the Brain Or IntestinaL EstimateD (BOILED-Egg) permeation model portrays 35DBTA8 as a poor candidate for a drug that would partition to the CNS (Fig. 7). Relatedly, we encountered problems with solubility of this compound in aqueous media; even its disodium salt (carboxylate and phenoxide) was poorly soluble, producing turbid solutions/suspensions. This rendered 35DBTA8 impractical for our applications and narrowed the choices down to 35DBTA7 and 35DBTSA12. It may be worth noting that very potent inhibitors such as 35DBTA3 can have drawbacks, including effects that approach a binary “on/off” manifestation and difficulties in achieving a scalable intermediate effect.

Brain Or IntestinaL EstimateD permeation method (BOILED-Egg) model portrays 35DBTA8 as a poor candidate for a drug that would partition to the CNS

The ultimate goal of these efforts is to treat human conditions having a neuroinflammatory component with SxC− inhibitors—if not alone, then as part of a combinatorial therapy. Several clinical trials in humans, which involved potential therapeutic agents have failed (especially in stroke), so this novel strategy seems worth investigating. The compounds identified here already serve as convenient molecular probes applicable in research on SxC− transport. Furthermore, the results of this study will guide the development of related optimized molecular tools useful for exploration of the SxC− pharmacophore. Neuroinflammation treatment in vivo studies utilizing a mouse model are in progress.