Immune Checkpoint blockers

Voltage-gated ion channels, which include potassium (KV), sodium (NaV), and calcium (CaV) channels, play crucial roles in the generation of action potentials and in synaptic transmission and are prime targets for photopharmacology. Not surprisingly, they have played a prominent role in the development of the field. In

2004, Trauner, Kramer, and Isacoff introduced a light and voltage sensitive potassium channel called SPARK.⁵⁹ To this end, they endowed a Shaker KV with an engineered cysteine, which served as a bioconjugation motif for a tethered quaternary ammonium ion (MAQ, Table 1, #30). Isomerization of the photoswitch with 380 nm/500 nm light shortened and lengthened the tether, unblocking and blocking the pore. This translated to the reversible optical control of action potential firing in hippocampal pyramidal neurons. An additional mutation in the channel pore converted the potassium-selective SPARK into a non-selective ion channel (D-SPARK).⁶⁰ While SPARK channels led to the optical of neuronal inhibition, the unselective variant enabled light-controlled depolarization and excitation of dissociated neurons.

Photocontrol over the mechanically gated potassium channel TREK1 was achieved by conjugating MAQ (Table 1, #31) with a TREK1 cysteine mutant.⁸¹ Isomerization with 380 nm/500 nm light resulted to reversible activation in HEK293T cells.

Transfecting hippocampal neurons with the TREK1 mutant and labelling with MAQ allowed insights in the formerly unknown role of TREK1 in GABAergic signaling.

While no light-elicited TREK1 currents could be detected at resting potential, baclofen-induced GABA currents could be modulated with light.

In addition to PCLs, cationic blockers that function as PTLs have been systematically explored (Fig. 4). Although these compounds are much less selective than MAQ as they affect a range of voltage gated ion channels, they have nevertheless proven to be powerful photopharmacological tools in vivo and they are on the verge of becoming clinically relevant. The first compound that functioned in this way was AAQ (Table1, #32).⁵⁵ Although it was initially designed to attach itself covalently via affinity labelling, it proved to be a PCL that binds to the inner cavity of voltage gated ion channels and has long-lasting effects to cells due to its lipophilicity.⁸² As such, it is a photoswitchable version of the well-known use-dependent open-channel blocker lidocaine. AAQ can be isomerized with 360 nm/500 nm light and is thermally relatively bistable. Suppression of AP firing could be achieved with 380 nm light, whereas 500 nm light restored neuronal activity. In a subsequent study, AAQ was used to convey light-sensitivity to blind retinae, where it mainly targets amacrine cells.⁸³ In addition to AAQ, a series of derivatives with different photochemical, thermal, and pharmacological profiles was synthesized and characterized in cells. Amongst these, the highly lipophilic switch PrAQ (Table1,

#33) turned out to be cis-active, inverting the logic of the otherwise trans-active blockers.

The photoswitches discussed above required UV irradiation for trans/cis isomerization and were thermally bistable, which limited their utility for applications in vision restoration. To address these issues, a second-generation of red-shifted photochromic ion channel blockers was developed that had absorption maxima in the 420–460 nm range.⁸⁴ The lead compounds that emerged from this study, DENAQ (Table 1, #34) and BENAQ (Table 1, #35) blocked K 3.1 and Shaker

potassium channels expressed in HEK293T cells in a light-dependent fashion and could be turned on and off with visible light and fast thermal relaxation, respectively (Fig. 4B and C). They worked in hippocampal brain slices and were subsequently used in vision restoration.^85–87 DENAQ provided sensitivity to white light at relatively low intensities and restored light-guided behavior in blind mice in an open field assay. Like DENAQ, BENAQ acted primarily on RGCs and showed long-lasting effects due to its high lipophilicity.

Figure 4: Photochromic ion channel blockers. (A) Schematic depiction of a photoswitchable, use-dependent open channel blocker (red star). Upon isomerization to its inactive form (pentagon), the blocker unbinds from the pore. (B) Light-dependent potassium current elicited by isomerization of DENAQ. Reprinted with permission from reference (84). Copyright 2011 American Chemical Society. (C) optical control of action potential firing in hippocampal neurons with PhENAQ.

Reprinted with permission from reference (84). Copyright 2011 American Chemical Society. (D)-(E) Multi electrode recordings in mouse retinae. Treatment with DAD results in light-dependent network activity (blue = 460 nm, black = dark).

Figure created by the authors from data in reference (94).

A symmetrically substituted azobenzene with a quaternary ammonium ion on each side, termed QAQ (Table 1, #36) was introduced shortly after its unsymmetrical predecessors.⁸⁸ QAQ responds to 380 nm/500 nm light, is stable in the dark and can block KV, NaV and CaV channels. This bivalent ion channel blocker is not membrane-permeable and needs to be actively transported into the cell via TRPV1 channels or P2X receptors. It was therefore possible to selectively target TRPV1 expressing cells for the optical control of nociception. More bistable and slightly red-shifted ortho-substituted derivatives of QAQ were reported shortly thereafter (Table 1, #37 and #38).⁸⁹ The even more red-shifted blocker QENAQ (Table 1, #39) was developed to combine the pharmacokinetics of QAQ with the photophysical

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properties of DENAQ.⁹⁰ Like QAQ, QENAQ blocks NaV and KV and can be used to optically control sensory DRG neurons.

The mechanism of action of DENAQ and BENAQ in degenerating retinae was unraveled in 2016 when Kramer and co-workers could show that they are subject to a similar transport mechanism as QAQ.⁸⁶ Like TRPV1, P2X receptors are known to dilate upon continued activation with ATP. As such, they can serve as an entrance gate in degenerating retinae. The authors suggested that this mechanism ensures that the photoswitch cannot be taken up into cells of a healthy retina, reducing side-effects. In vivo administration of these compounds requires intraocular injection, which is a standard procedure in ophthalmology, but carries a residual infection risk. Therefore, the slow release of QAQ and DENAQ from polymeric matrices was investigated.⁹¹

The permanently charged photoswitchable trimethylammonium ion AzoTAB (Table1, #40) had been used as a photoswitch in material science before it was developed into as photochromic ion channel blocker.⁹² AzoTAB is isomerized from trans to cis with UV light and can be switched back with blue light. In cardiomyocytes, the trans isomer suppressed spontaneous electric excitability. The shape of action potentials indicated a trans block of fast NaV and CaV channels. In more recent work, AzoTAB was shown to potentiate KV currents in the dark.⁹³ Further studies culminated in the development of third-generation open-channel blockers, such as DAD (Table 1, #41).⁹⁴ In contrast to all previously described photoswitches, DAD does not bear a permanent charge and can rapidly diffuse across biological barriers in tissues. DAD can be isomerized with blue or white light and relaxes quickly in the dark. It mainly acts on bipolar cells exploiting the extant circuitry of a blind retina (Fig. 4D and E).

The photochromic blocker fotocaine (Table 1, #42),⁹⁵ another compound that is not permanently charged, was developed through “azologization” of the analgesic fomocaine. Azologization refers to the systematic replacement of isosteric motifs in drugs with azobenzenes.⁹⁶ Fotocaine can be isomerized with 350 nm/450 nm light, allowing and suppressing action potential firing in dissociated mouse hippocampal neurons.

In 2017, Berger and co-workers demonstrated that open-channel blocking of voltage-gated ion channels can also be achieved with guanidinium ions.⁹⁷ They synthesized an azobenzene version of the known open-channel blocker 2GBI, photoGBI (Table 1, #43) to inhibit the voltage-gated proton channel HV1. The trans-active blocker can be removed from the pore by illumination with blue light to induce proton currents in macrophages and sperm.

Other Ion Channels