SUMMARYObjective: Brivaracetam (BRV) decreases seizure activity in a number of epilepsy models and binds to the synaptic vesicle glycoprotein 2A (SV2A) with a higher affinity than the antiepileptic drug levetiracetam (LEV). Experiments were performed to determine if BRV acted similarly to LEV to induce or augment short-term depression (STD) under high-frequency neuronal stimulation and slow synaptic vesicle recycling. Methods: Electrophysiologic field excitatory postsynaptic potential (fEPSP) recordings were made from CA1 synapses in rat hippocampal slices loaded with BRV or LEV during intrinsic activity or with BRV actively loaded during hypertonic stimulation. STD was examined in response to 5 or 40 Hz stimulus trains. Presynaptic release of FM1-43 was visualized using two-photon microscopy to assess drug effects upon synaptic vesicle mobilization. Results: When hippocampal slices were incubated in 0.1-30 lM BRV or 30 lM-1 mM LEV for 3 h, the relative CA1 field EPSPs decreased over the course of a high-frequency train of stimuli more than for control slices. This STD was frequency-and concentrationdependent, with BRV being 100-fold more potent than LEV. The extent of STD depended on the length of the incubation time for both drugs. Pretreatment with LEV occluded the effects of BRV. Repeated hypertonic sucrose treatments and train stimulation successfully unloaded BRV from recycling vesicles and reversed BRVs effects on STD, as previously reported for LEV. At their maximal concentrations, BRV slowed FM1-43 release to a greater extent than in slices loaded with LEV during prolonged stimulation. Significance: BRV, similar to LEV, entered into recycling synaptic vesicles and produced a frequency-dependent decrement of synaptic transmission at 100-fold lower concentrations than LEV. In addition, BRV slowed synaptic vesicle mobilization more effectively than LEV, suggesting that these drugs may modify multiple functions of the synaptic vesicle protein SV2A to curb synaptic transmission and limit epileptic activity. KEY WORDS: Antiepileptic drugs, Anticonvulsants, Epilepsy, Brivaracetam, Levetiracetam, SV2A, Synaptic vesicle recycling, Short-term depression.Brivaracetam (BRV) is an antiepileptic drug that recently showed positive outcome in phase III clinical trials for treatment of partial-onset seizures.1 BRV and the closely related compounds levetiracetam (LEV) and seletracetam (SEL) bind to synaptic vesicle glycoprotein 2A (SV2A), with BRV affinity about 20-fold greater than LEV.2,3 Several mechanisms have been proposed for the antiepileptic action of LEV, including interference with SV2A or blocking of calcium currents. 4 In contrast, BRV does not bind to calcium channels and its effect on sodium channels does not alter sustained activity.3,5

Human cytomegalovirus (HCMV) terminase complex entails a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We report herein the design, synthesis, and characterization of dihydroxypyrimidine (DHP) acid ( 14), methyl ester (13), and amide (15) subtypes as inhibitors of HCMV pUL89-C. All analogs synthesized were tested in an endonuclease assay and a thermal shift assay (TSA) and subjected to molecular docking to predict binding affinity. Although analogs inhibiting pUL89-C in the sub-μM range were identified from all three subtypes, acids ( 14) showed better overall potency, substantially larger thermal shift, and considerably better docking scores than esters (13) and amides (15). In the cell-based antiviral assay, six analogs inhibited HCMV with moderate activities (EC 50 = 14.4−22.8 μM). The acid subtype (14) showed good in vitro ADME properties, except for poor permeability. Overall, our data support the DHP acid subtype (14) as a valuable scaffold for developing antivirals targeting HCMV pUL89-C.

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