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. 2023 Mar 6;30(1):16.
doi: 10.1186/s12929-023-00907-5.

Novel, thalidomide-like, non-cereblon binding drug tetrafluorobornylphthalimide mitigates inflammation and brain injury

Daniela Lecca  1 , Shih-Chang Hsueh  1 , Weiming Luo  1 , David Tweedie  1 , Dong Seok Kim  2   3 , Abdul Mannan Baig  4 , Neil Vargesson  5 , Yu Kyung Kim  3 , Inho Hwang  3 , Sun Kim  3 , Barry J Hoffer  6 , Yung-Hsiao Chiang  7   8 , Nigel H Greig  9
Affiliations

Novel, thalidomide-like, non-cereblon binding drug tetrafluorobornylphthalimide mitigates inflammation and brain injury

Daniela Lecca et al. J Biomed Sci. .

Abstract

Background: Quelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurological disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs.

Methods: TFBP/TFNBP were synthesized and evaluated for cereblon binding and anti-inflammatory actions in human and rodent cell cultures. Teratogenic potential was assessed in chicken embryos, and in vivo anti-inflammatory actions in rodents challenged with either lipopolysaccharide (LPS) or controlled cortical impact (CCI) moderate traumatic brain injury (TBI). Molecular modeling was performed to provide insight into drug/cereblon binding interactions.

Results: TFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with LPS, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 h post-injury following CCI TBI. Compared to vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2-weeks post-injury. Behavioral evaluations at 1- and 2-weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice.

Conclusion: TFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurological disorders involving a neuroinflammatory component.

Keywords: Cereblon; Immunomodulatory imide drugs (IMiDs); Microglia; Neurodegeneration; Neuroinflammation; Spalt like transcription factor 4 (SALL4); Teratogenicity; Thalidomide.

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Conflict of interest statement

TFBP and TFNBP are protected under US Patent Application No. 63/397,235 2022. NHG, WL, DL, DT are coinventors and have assigned their rights in entirety to NIA, NIH (i.e., the US Government). DSK is, likewise, a co-inventor and has assigned his rights to AevisBio Inc. NIA, NIH and AevisBio Inc., have a Cooperative Research and Development Agreement to develop novel thalidomide-like drugs for the treatment of neurological disorders involving excessive inflammation. DSK, YKK, IH, SK are employees and shareholders of AevisBio Inc. All other authors declare no conflict of interest. The funders had no role in the design of the studies; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Fig. 1
Fig. 1
TFBP and TFNBP do not bind cereblon or lower levels of neo-substrate SALL4. Chemical structures of thalidomide (A) and pomalidomide (B). The binding of TFBP (C) and TFNBP (D) to cereblon was examined through use of a cereblon/BRD3 binding FRET assay (E). TFBP and TFNBP were not able to bind cereblon (IC50 53.45 μM and > 100 μM, respectively), compared to Pom (IC50 3.36 μM) (F). Concentration- dependent degradation of the downstream neo-substrates SALL4 was evaluated in human Tera-1 cells. TFBP and TFNBP at 0.1 μM and 1 μM did not lower expression levels of SALL4, in contrast to Pom at a 1 μM concentration (G, H). *p < 0.05 vs. control group. Values are presented as mean ± S.E.M., of n observations [N = 2 or 3 per group over 8 concentrations (E) to generate an IC50 value (F); N = 3 per group re: SALL4 expression (H)]
Fig. 2
Fig. 2
TFBP, TFNBP and IMiD drug docking pockets in chain C of human cereblon with the binding scores. (A) Determination of pockets for IMiD interactions within chain C of human cereblon showed the top 3 pharmacophores with their attributes (A1), with the pocket #1 (A, B, yellow circles) for binding the classic IMiDs thalidomide and pomalidomide (B, B1, B2). (C) The compounds TFNBP and TFBP docked with a lesser predicted preference at pocket #1 with scores (C-arrows and green strips) that demonstrated a substantially lower affinity than thalidomide and pomalidomide, which docked at pocket #1 with greatest preference and a higher binding affinity and associated Vina scores (B-green strips in insets). The binding pocket preferences and Vina scores of TFNBP and TFBP reflect a poor interaction probability of these compounds within the classic thalidomide binding pharmacophore (pocket #1)
Fig. 3
Fig. 3
TFBP and TFNBP mitigate LPS-induced increases of nitrite and TNF-α in RAW 264.7 cell cultures. Challenge with LPS induced a spike in levels of nitrite and TNF-α, as compared to non-treated control cells (data not shown). Administration of TFBP reduced nitrite expression at the lowest evaluated 100 nM concentration (B), whereas elevated levels of TNF-α were mitigated starting at 600 nM (C). TFBP was well tolerated and was without impact on cell viability (A). Treatment with TFNBP was likewise effective in decreasing levels of nitrite and TNF-α, without affecting cell viability (Fig. 2D–F). *p < 0.05; **p < 0.01; ****p < 0.0001 vs. cnt-dmso group. N = 4 per group
Fig. 4
Fig. 4
TFBP and TFNBP significantly decreased levels of TNF-α, IFN-γ and IL-5 in plasma and cortex of LPS- challenged animals. TFBP and its analogue TFNBP were evaluated in a LPS model of inflammation in rats (LPS 1 mg/ kg, i.p.). In this model, systemic administration of LPS induces elevations in pro-inflammatory proteins at 4 h in both plasma and brain (cerebral cortex). TFBP (formulated as a suspension in carboxymethyl cellulose (CMC) and administered i.p.) significantly decreased levels of pro-inflammatory cytokines (particularly TNF-α and IL-5) in both plasma and cortex, more significantly than TFNBP (A, B; E, F, respectively). A post-treatment reduction of IFN-γ was observed in plasma but not in cerebral cortex (C, D) On this basis, TFBP was selected for further in vivo investigation. *p < .05, **p < .01, ****p < .0001 vs saline control group. #p < .05, ##p < .01, ###p < .001, ####p < .0001 vs LPS-treated group. ‘N value of animals’ shown at the base of each bar within brackets
Fig. 5
Fig. 5
TFBP partially improved motor functions after TBI. In the beam walking test, CCI- challenged mice showed an increase in average time needed to traverse the beam (A), as well as in immobility time spent on the beam (B). TFBP (16.25 mg/kg and 32.5 mg/kg, i.p.), especially at the highest tested dose, mitigated this injury-induced increase, as seen in the behavioral assessment performed after 1 week. (A, B). Gait analysis was performed through DiGi Gait System (Mouse Specifics, Inc.). Treatment with TFBP (HD, high dose) countered the TBI-induced increase in brake time (time between initial paw contact to maximum paw contact), 1 week post injury (C). *p < 0.05, ***p < 0.001 vs control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs CCI group). ‘N value of animals’ shown at the base of each bar within brackets
Fig. 6
Fig. 6
TFBP significantly decreased cortical lesion volume in CCI-challenged mice. CCI animals show loss of cortical tissue near the lesion site (A), as well as an enlargement of the lateral ventricle size. Treatment with TFBP (16.25 mg/kg and 32.5 mg/kg, i.p.) significantly reduced lesion volume induced by TBI; a similar trend is noticeable for the lateral ventricle size, although this does not reach statistical significance (B). Representative images of Giemsa-stained cortical sections (C). *p < 0.05, ****p < 0.0001 vs control; #p < 0.05 vs CCI). ‘N value of animals’ shown at the base of each bar within brackets
Fig. 7
Fig. 7
TFBP mitigates TBI-mediated expression of activation of microglial cells. CCI induces morphological changes in microglial cells (A), which are representative of an activated phenotype. Multiple parameters of Iba1 + cell morphology were analyzed, including ramification index (B), spanned area (C), number of branches (D), junctions (E) and endpoints (F). TFBP (16.25 mg/kg and 32.5 mg/kg, i.p.) mitigated the morphological changes induced by CCI in cerebral cortex, evaluated at 2 weeks post-injury (B–F). Representative images of Iba1 + cells at × 40 magnification and their skeleton reconstruction through MotiQ software (A). *p < 0.05, ****p < 0.0001 vs control group; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs CCI group; + + + + p < 0.0001 vs Contralateral side. ‘N value of animals’ shown at the base of each bar within brackets
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