Altiratinib

Dear Editor, This study aimed to develop topical therapeutics for the treatment of UV-induced skin hyperpigmentary disorders and demonstrates how a compound identified through a robust mechanism-based screen was successfully engineered using medicinal chemistry. CREB-regulated transcription co-activator 3 (CRTC3) is the critical upstream regulator of MITF, which is a central regulator of melanogenesis by modulating tyrosinase, tyrosinase-related protein-1 (TYRP1), and dopamine tautomerase (DCT) in ultraviolet (UV) or cyclic adenosine monophosphate that is forskolin (FSK)-induced pathways.1-3 Building upon the discovery of a tunable and reversible regulation of MITF by CRTC3 nuclear shuttle,4 we established a screening tool for CRTC3 inhibitors and performed a high-throughput screening of small molecules targeting CRTC3. While altiratinib (ALT) was screened and demonstrated a dose-dependent inhibitory action on CRTC3 activity and melanogenesis up to 1 µM, cytotoxicity emerged at higher concentrations (Figure S1A). There is a need to enhance both its safety margin and efficacy to address the limitations of anti-pigmentation topical drugs or cosmeceuticals.5 Since, ALT was developed to target multiple-growth factor receptors, c-Met, VEGFR2 and TrKA,6 we explored the melanin-inhibiting properties of other functionally comparable inhibitors, including sorafenib, foretinib and cabozantinib. While sorafenib was the only compound that exhibited a dose-dependent reduction in melanin content in mouse melanocytes (Mel-Ab), its efficacy was inferior to that of ALT. Furthermore, all showed cytotoxicity in Mel-Ab and did not have anti-melanogenesis effects on normal human melanocytes (NHM) with more pronounced cytotoxicity (Figure S1B–E). To heighten potency, safety and skin barrier permeability for human skin application,7 we designed new compounds using a bioisosteric replacement strategy that incorporated different ring systems of ALT.8 We selected representative analogues from each bicyclic group (6, indazole; 7, benzoxazole; 8, benzothiazole) while considering their cytotoxicities and potencies, resulting in the creation of a series of ALT analogues (Figure S2A–D). Subsequently, we screened ALT analogues that exhibited no more than 10% cytotoxicity at concentrations ten times higher than their effective concentration and a concentration-dependent melanin-inhibiting effect in both Mel-Ab and B16F10 mouse melanoma cells. Through this process, we identified ALT6a, ALT7a, and ALT8c which demonstrated comparable to or better than ALT in inhibition of FSK-stimulated melanogenesis without cytotoxicity (Figures S3A,B and S4A,B). Generally, NHM exhibited higher sensitivity than Mel-Ab to cytotoxicity at a concentration of 10 µM, (Figure 1A). ALT6a and ALT7a consistently demonstrated anti-melanogenesis activity, while ALT8c, which reduced melanin content in Mel-Ab and B16F10, did not inhibit melanogenesis in NHM (Figure 1B) positioning ALT7a as top and ALT6a as second top candidates that exert a dose-dependent reduction in melanin content with no or minimal cytotoxicity, respectively, in all three cells (Figure 1A,B and Figures S3 and S4). ALT6a and ALT7a demonstrated superior efficacy and a wider safety margin to ALT, although the safety margin of ALT6a was slightly narrower than that of ALT7a in NHM (Figure 2A). In NHM, ALT6a and ALT7a displayed a more efficient reduction in melanin content than ALT (Figure 2B), whereas the efficacy of melanin reduction achieved by ALT6a and ALT7a was comparable to that observed with ALT in Mel-Ab and primary melanocytes cultured from KRT14-stem cell factor (SCF)-epidermal humanized mice9 (Figure 2C and Figure S7A–C). ALT, ALT6a and ALT7a for 72 h resulted in a comparable decrease in the FSK-induced elevation of cellular tyrosinase activity in Mel-Ab (Figure 2D), whereas in tube mushroom tyrosinase activity (Figure 2E) and 2 h-short-term cellular tyrosinase activity remained unaffected (Figure S6). Then, we investigated whether the decreased melanin content and cellular tyrosinase activity by ALT6a and ALT7a were linked to a reduction of pigmentation genes. 72 h of FSK-stimulated upregulation of mRNA and protein levels of melanogenesis genes were significantly attenuated by pretreatment with ALT or ALT6a and ALT7a (Figure 2F,G), whereas short-term treatment (up to 8 hours) with these compounds selectively inhibited mRNA and protein expression levels of MITF, but not those of tyrosinase, TYRP1, and DCT (Figure 2H,I). Mechanistically, they suppressed the FSK-stimulated transcriptional activity of CREB (Figure 2J), which was attributed to the phosphorylation-dependent intracellular localization of CRTC3 by ALT6a and ALT7a (Figure 2K–M and Figure S7). Exploring relevant kinase profiles suggests that AMPK and ERK are presumed to be responsible for CRTC3 hyperphosphorylation mediated by ALT, ALT7a and ALT6a (Figure 2K and Figure S8). Interestingly, while ALT downregulated JNK and AKT activity, ALT7a and ALT6a did not affect them, potentially contributing to the differential effects on efficacy and cell viability between ALT and ALT7a or ALT6a (Figure S8). To evaluate the potential of ALT6a and ALT7a as skin topicals, a skin parallel artificial membrane permeability assay (PAMPA) was performed.10 In comparison to ALT, ALT6a and ALT7a exhibited enhanced skin permeability (Figure 3A). We further assessed the efficacy and skin tissue toxicity of ALT 6a and ALT7a in KRT14-SCF mice and UVB-irradiated human skin explants. The topical application of ALT6a and ALT7a to the tails of KRT14-SCF mice more potently reduced epidermal melanin (Figure 3B–D) and tyrosinase protein levels (Figure 3E) than ALT. In UVB-irradiated human skin culture for 24 hours, UVB triggered the nuclear translocation of CRTC3 in epidermal melanocytes, which was attenuated by topical exposure to ALT, ALT6a and ALT7a (Figure 3F). UVB-stimulated melanin deposition (Figure 3G,H) and upregulation of melanogenesis gene expression were more potently reduced by ALT6a and ALT7a (Figure 3I) than by ALT. This suggests that the enhanced anti-pigmentation potency observed in mouse skin and human skin for ALT6a and ALT7a, especially at lower doses, may likely be attributed to their improved skin barrier permeability due to modifications in the chemical structure. These findings highlight the potential of refining topical drugs with enhanced skin permeability alongside efficacy and safety through medicinal chemistry. Numerous skin-lightening topicals targeting tyrosinase have been developed but none were safe and efficacious enough.2, 3, 5 As our previous studies demonstrated,1 targeting CRTC3 can attain the key treatment goal of hyperpigmentation disorders, not the death of melanocytes but the homeostatic modulation of melanin synthesis.5 We showed that ALT6a and ALT7a serve as excellent examples for drug repositioning and small-molecule fabrication, highlighting their potential for developing highly potent and non-toxic topicals for the treatment of extremely common UV and photo-ageing-associated hyperpigmentary diseases. Conceptualization and design of the experiments, Hongchan An, Sung Eun Chang and Youngsup Song; methodology, Jeong Hyeon Lee, Hongchan An, Hye Ji Kwon, Seung Jin Lee, Hayoung Hwang, Sung Eun Chang and Youngsup Song; investigation and visualization and curation of the data, Jeong Hyeon Lee, Hye Ji Kwon, Seung Jin Lee, Young Hye Park, Ji sun Hwang, Min Young Kim, Hayoung Hwang, JeongYoon Kim and Seung Jin Lee; supervision of the experiment, Hongchan An, Sung Eun Chang and Youngsup Song; Writing the original draft, Jeong Hyeon Lee, Hongchan An, Sung Eun Chang and Youngsup Song; Edition of the manuscript, Sung Eun Chang and Youngsup Song. We would like to thank Dr. Jin Sup Eom in the AMC Plastic Surgery Department for providing human skin samples and Asan Life Science Institute and Asan Medical Center (AMC) Pathology Laboratory for the tissue-processing support. The authors declare no conflict of interest. This study was supported by a grant from the National Research Foundation of Korea (2020R1A4A4079708, RS-2023-00208426, RS-2023-00246165) and Ministry of Health & Welfare, Republic of Korea (HP23C0072). The utilization of human skin tissue for ex vivo research was granted approval by the Institutional Review Board (IRB) of Asan Medical Center (IRB no. 2020-0091). This human skin tissue was obtained from individuals who had been informed, provided voluntary consent, and had undergone neck or abdomen reduction surgery at Asan Medical Center under the same IRB number (2020-0091). All animal studies were conducted according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC no 2020-02-248) of the Asan Medical Center, Seoul, Korea. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii , Plasmodium , and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii . We have identified Tg PRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for Tg PRP4K and for its Plasmodium falciparum counterpart, Pf CLK3. Our data identified structural features critical for binding of the other Pf CLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum . Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.