*3.10. Molecular Docking*

Molecular docking studies were performed using MOE 2016. The crystal structure of human *α*4*β*2 nAChR (PDB identifier: 5kxi) was obtained from the Protein Data Bank (http://www.rcsb.org, accessed on 28 September 2016). Prior to docking, heteroatoms and water molecules in the protein were removed. In the meantime, compounds were minimized. The result of each ligand was furtherly analyzed by using PyMOL.

#### **4. Conclusions and Discussion**

In conclusion, the genus *Pseudogymnoascus* is a group of psychrophilic fungi that possess good potential in serving structurally unique structures but not widely investigated. In this study, chemical investigation of secondary metabolites from the psychrophilic fungus *Pseudogymnoascus* sp. HDN17-933 led to the discovery of six new tetrapeptides psegynamides A–F (**1**–**6**), among which psegynamides D–F (**4**–**6**) was the first naturally occurring peptide bearing a tetrahydropyridoindoles moiety. To the best of our knowledge, only a few synthetic peptides bearing the tetrahydropyridoindoles motif have been reported [30,31], for example, tetrahydropyridoindoles as cholecystokinin and gastrin antagonists in 1992 [30]. Solid synthesis techniques assisted by Marfey's method were

employed to work out the absolute configuration, especially in solving the challenges of determining the order of *L*-Val and *D*-Val isomers in the peptide sequences. Moreover, compound **2** was found to have bioactivity by inhibiting human nAChRs. Considering that non-steroidal anti-inflammatory drugs and narcotics (opioids) are currently the most commonly used analgesic drugs, these drugs exhibit limitations in efficacy, unwanted side effects, and the problem of drug abuse. To overcome these problems, the discovery of different molecular participants in the pain pathways could bring new opportunities for therapeutic intervention. Compound **2** might be helpful in developing potential natural short peptide inhibitor of nAChRs from Antarctica-derived fungus. The above findings illustrate and highlight the validity of exploiting psychrophilic fungus for the discovery of structurally novel and bioactive natural products.

**Supplementary Materials:** The following are available as supplementary materials https://www. mdpi.com/article/10.3390/md20100593/s1: Figure S1: 18S rRNA sequences data of *Pseudogymnoascus* sp. HDN17-933; Figure S2: HPLC analysis of the crude extract of HDN17-933; Figures S3–S57: 1D and 2D NMR spectra, HRESIMS spectra, IR spectra of compounds **1**–**6**; Figures S58–S88: Advanced Marfey's acid/alkaline hydrolytic analysis of compounds **1**–**6**, and NMR spectra of solid-phase total synthesis. Table S1. Table of compounds (**1**–**6**) inhibition of ACh-evoked peak current amplitude mediated by human (h) α1β1εδ, α1β1γδ, α3β2, α3β4, α4β2, α7 and α9α10 nAChRs. Table S2. Free binding energy estimation and molecular interactions of compounds **1**–**6.**

**Author Contributions:** The contributions of the respective authors are as follows: X.H.: conceptualization, resources, investigation, writing—original draft, and writing—review and editing. C.L., R.Z., Y.L., H.L. and Y.Z.: investigation and data curation. H.-S.T. and R.Y.: assistance of nicotinic acetylcholine receptors activity and molecular docking. D.L., G.Z., T.Z. and Q.C.: funding acquisition, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (2022QNLM030003-1, 2022QNLM030003-2), National Natural Science Foundation of China (81991522, 41976105), the NSFC-Shandong Joint Fund (U1906212), the Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (2021CXLH0012), Taishan Scholar Youth Expert Program in Shandong Province (tsqn 202103153, tsqn 201812021), Major Basic Research Programs of Natural Science Foundation of Shandong Province (ZR2019ZD28) and an Australian Research Council (ARC) Discovery Project Grant (DP150103990) awarded to D.J. Adams.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Adams for the facilities and support of the functional studies carried out in *Xenopus* oocytes at IHMRI, University of Wollongong.

**Conflicts of Interest:** The authors declare no conflict of interest.
