**1. Introduction**

Psychrophilic fungi are a group of cold-adapted fungi residing in polar regions, alpine permafrost, glaciers, deep oceans, and other habitats [1–6], which are known for long-term low temperature, strong ultraviolet radiation, low nutrient and water availability, and frequent freeze and thaw cycles [7–9]. In order to adapt to such harsh conditions, these fungi have evolved special strategies in their metabolism and physiology [7,10–12], which endows the ability to produce diversified secondary metabolites, and makes psychrophilic fungi competitive microorganism group to serve structurally novel and bioactive natural products for drug development [13–16].

So far, a large number of novel natural products have been isolated from psychrophilic fungi, such as trisorbicillone A, a novel sorbicillin trimer that showed cytotoxic against HL60 cell lines (IC50 3.14 μM) from deep-sea fungal strain *Phialocephala* sp. [17]; brevione A, the first breviane spiroterpenoid family from *Penicillium brevicompactum* [18]; penilactones

**Citation:** Hou, X.; Li, C.; Zhang, R.; Li, Y.; Li, H.; Zhang, Y.; Tae, H.-S.; Yu, R.; Che, Q.; Zhu, T.; et al. Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic *Pseudogymnoascus* sp. HDN17-933. *Mar. Drugs* **2022**, *20*, 593. https:// doi.org/10.3390/md20100593

Academic Editor: Rosa Maria Vitale

Received: 26 August 2022 Accepted: 19 September 2022 Published: 22 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

A and B, novel polyketides with NF-kB inhibitory activity (inhibitory rate of 40% at 10 Mm) from *Penicillium crustosum* PRB-2 [19]; 16-membered trichobotryside A inhibiting the larvae settlement of Balamus amphitrite (EC50 2.5 μg/mL) and 18-membered trychobotrysides B-C from *Trichobotrys effuse* [20]; and cytotoxic diterpenes conidiogenones B–G isolated from *Penicillium* sp, and conidiogenone C displayed significant cytotoxicity against HL60 and BEL-7047 cell lines with IC50 values of 0.038 mM and 0.9 mM respectively [21].

During our ongoing research on searching bioactive structures from Antarctic-derived fungi, a psychrophilic strain *Pseudogymnoascus* sp. HDN17-933, isolated from sand samples collected from Fildes Peninsula, was chosen for study based on its unique HPLC-UV profile. Detailed chemical investigation on its fermentation products afforded six tetrapeptides psegynamides A–F with human nicotinic acetylcholine receptors (nAChRs) targeting activity. Among those structures, psegynamides D–F (**4**–**6**) represented the first group of naturally occurring tetrapeptides carrying tetrahydropyridoindole units in the backbones. Herein, we will describe the isolation, structural elucidation, solid-phase total synthesis, and biological activity evaluation of these new compounds.

#### **2. Results and Discussion**

The psychrophilic fungal strain *Pseudogymnoascus* sp. HDN17-933 was isolated from Antarctica. They can grow in an environment of −5 ◦C, and the optimum growth temperature is 15–18 ◦C. The studies of their secondary metabolites are infrequent, and only 44 secondary metabolites have been reported until the time of writing. In this paper, the strain was cultured under static conditions at 15 ◦C for 30 days in a rice medium. The EtOAc extract of strain *Pseudogymnoascus* sp. HDN17-933 (Figure S2) was fractionated and purified by vacuum chromatography on silica gel, octadecyl-silica (ODS), Sephadex LH-20, and HPLC to afford six tetrapeptides psegynamides A–F (**1**–**6**) (Figure 1).

**Figure 1.** The structures of compounds **1**–**6** isolated from *Pseudogymnoascus* sp. HDN17-933.

Compound **1** named psegynamide A was obtained as a white amorphous powder. Its molecular formula was determined to be C30H39N5O5 based on the molecular ion peak at *m/z* 550.3025 [M + H]+ (calcd 550.3024) in the HRESIMS analysis, indicating 14 degrees of hydrogen deficiency. In the 1H-NMR spectrum of **1** (Table 1), the characteristic signals of NH were displayed at low-field *δ*<sup>H</sup> 8.68 (1H, d, *J* = 9.5 Hz), 8.37 (1H, d, *J* = 7.8 Hz), and 8.13 (1H, d, *J* = 9.1 Hz). The signals of *α*-proton, the characteristic for amino acid residues, were exhibited at *δ*<sup>H</sup> 4.56 (1H, m), 4.44 (1H, m), 4.30 (1H, m), and 4.18 (1H, m). Four methyl groups were displayed at *δ*<sup>H</sup> 0.72 (3H, d, *J* = 6.8 Hz), 0.68 (3H, d, *J* = 6.8 Hz), 0.61 (3H, d, *J* = 6.8 Hz), and 0.48 (3H, d, *J* = 6.8 Hz). Analysis of the 13C-NMR and HSQC spectra indicated the presence of four carbonyl peaks assignable to amide carbonyl groups at *δ*<sup>C</sup> 173.6, 171.1, 170.7, and 161.9. Four groups of *α*-proton signals were showed at *δ*<sup>C</sup> 57.3, 57.1, 53.9, and 52.9. The appearance of these spectra was typical of peptides. Combined

analyses of 1H-1H COSY, HMBC, HSQC, NOESY, and TOCSY spectra further revealed the presence of 1 × Trp, 2 × Val, and 1 × Phe residues (Table 1), which were in accordance with the result of Marfey's analysis (Figure S58). In the HMBC spectrum (Figure 2), NH of Val (1) has a correlation signal with C-1 on Trp; NH of Val (2) has a correlation signal with C-1 on Val (1); NH of Phe has a correlation signal with C-1 on Val (2). Meanwhile, in the NOESY spectrum, the cross-peaks of NH in Val (1) and H-2 in Trp, NH in Val (2) and H-2 in Val (1), NH in Phe and H-2 in Val (2) further confirmed the linkage of amino acid residues by HMBC spectrum. Thus, the planar structure of compound **1** was established as Trp1-Val2-Val3-Phe4.

**Figure 2.** Key COSY, HMBC, and NOESY correlations of **1**–**6**.

The advanced Marfey's acidolytic method was considered to determine the absolute configurations of compound **1** [22]. After FDAA derivatization and assisted by HPLC analysis, the presence of *L*-Val, *D*-Val, and *D*-Phe in compound **1** (Figure S58) was confirmed. However, due to the destruction of Trp residue during the strong acid environment, we failed to detect the Trp-FDAA derivative from the hydrolytic mixture. Therefore, we turned to using alkaline hydrolytic to determine the absolute configuration of Trp residue. With the alkaline hydrolytic condition of 5 M LiOH at 110 ◦C for 16 h was finally adopted, compound **1** was successfully hydrolyzed, and the absolute configuration of Trp was determined as *L*. As analyzed from the HPLC profile, both *L*-Val and *D*-Val derivatives were detected in the acidolytic mixture of compound **1**, which makes it another challenge to determine the accurate sequence of the tetrapeptide. Hence, two possible stereoisomers, **1a** (*L*-Trp1-*L*-Val2-*D*-Val3-*D*-Phe4) and **1b** (*L*-Trp1-*D*-Val2-*L*-Val3-*D*-Phe4), were prepared using solid-phase total synthesis (Scheme 1A). By comparing the experimental NMR spectrum with the synthesized NMR data, the absolute configuration of compound **1** (Scheme 1B, Figure S64) was determined to be *L*-Trp1-*D*-Val2-*L*-Val3-*D*-Phe4, which was inconsistent with the synthesized stereoisomer **1b**.




**Scheme 1.** (**A**): Solid-Phase Synthesis of **1a**, **1b** and **2a, 2b**. (**B**): Comparison of 1H-NMR spectra of authentic **1,** synthetic **1a,** and **1b**.

Compound **2** named psegynamide B was obtained as a white amorphous powder. Its molecular formula was determined to be C31H41N5O5 based on the molecular ion peak at *m/z* 564.3172 [M + H]<sup>+</sup> (calcd 564.3180) in the HRESIMS analysis, which showed 14 Da molecular weight (MW) surplus to compound **1**. The 1H and 13C NMR of **2** were

similar to those of **1** (Table 1), except for the presence of a methoxy group. The 1H, 13C NMR, and HSQC spectra showed a signal at *δ*<sup>C</sup> 52.4/*δ*<sup>H</sup> 3.56, which was assignable to a methoxy group. In addition, the methoxy site was established unambiguously by an HMBC experiment, in which a long-range correlation between CH3 (*δ*<sup>H</sup> 3.56) and C-1 (*δ*<sup>C</sup> 172.6) in the Phe unit was observed. Combined analyses of 1H-1H COSY, HMBC, HSQC, and TOCSY spectra assigned the linkage of amino acid residues as Trp1-Val2-Val3- Phe4-OCH3. Marfey's acidolytic analysis confirmed the presence of *L*-Val, *D*-Val, and *D*-Phe in compound **2**, which was a similar case to compound **1**. Then, the solid-phase total synthesis and Marfey's alkaline hydrolytic method were also used to determine the absolute configurations of compound **2**. Finally, the absolute configuration of **2** was assigned to be *L*-Trp1-*D*-Val2-*L*-Val3-*D*-Phe4-OCH3.

Compound **3** named psegynamide C was isolated as a white amorphous powder. The HRESIMS analysis of compound **3** gave a hydrogen adduct [M + H]+ at m/z 566.2977 (calcd 566.2973), corresponding to the molecular formula of C30H39N5O6, which showed one more oxygen than compound **1**. In the 1H NMR spectrum (Table 1), two aromatic H-atom signals appearing at *δ*<sup>H</sup> 6.97 (2H, overlap, m) and 6.59 (2H, overlap, m) were attributable to a 1,4-substituted phenyl. In addition, a characteristic signal of OH was observed at low-field *δ*<sup>H</sup> 9.18 (1H, s). Careful analysis of NMR of compound **3** revealed a similar structure as compound **1**, except for the presence of Tyr residue instead of Phe residue. Thus, the planar structure of compound **3** was established as Trp1-Val2-Val3-Tyr4. Based on Marfey's acid/alkaline hydrolytic analysis, as well as solid-phase total synthesis, the absolute configuration of **3** was assigned to be *L*-Trp1- *D*-Val2-*L*-Val3-*D*-Tyr4.

Compound **4** was obtained as a white amorphous powder. Its molecular formula was determined to be C31H39N5O5 on the basis of the molecular ion peak at *m/z* 562.3032 [M + H]+ (calcd 562.3024) in the HRESIMS analysis, indicating 15 degrees of hydrogen deficiency. Compared the 1D-NMR data with compound **1**, compound **4** has an additional methylene (*δ*<sup>C</sup> 40.8) and the CH-5 of Trp in compound **1** was changed as a quaternary carbon. In addition, one more degree of unsaturation indicated a cyclic structure for compound **4**. With the assistance of COSY correlations of H2-5 /NH-2 , H-2/NH-2 and H-2/H2-3, and the HMBC correlations from H2-5 to C-4/C-2, the tetrahydropyridoindole residue was deduced. Compound **4** represented the first example naturally occurring peptide with a tetrahydropyridoindole moiety. On the basis of Marfey's acid hydrolytic analysis and solid-phase total synthesis, the absolute configuration of **4** was assigned to be as alternating LDLD chirality and named psegynamide D.

Psegymamides E-F (**5**–**6**) were obtained as white amorphous powders with the molecular formulas of C32H41N5O5 and C32H41N5O6 by HRESIMS, respectively. The 1D NMR spectra of **5** and **6** (Table 2) indicated a skeleton similar to psegynamide D (**4**). The difference between **4** and **5** was the replacement of the hydroxide group by a methoxy group (*δ*<sup>C</sup> 52.3/*δ*<sup>H</sup> 3.57) in the Phe unit, which was confirmed by HMBC correlation from OCH3 (*δ*<sup>H</sup> 3.57) and C-1 (*δ*<sup>C</sup> 172.7). The difference between **4** and **6** was the presence of Tyr-OCH3 residue instead of Phe residue. Thus, the planar structures of **5**–**6** were established. Similarly, the absolute configuration of **5**–**6** was assigned as alternating LDLD chirality by Marfey's method and solid-phase total synthesis and finally named psegynamides E-F, respectively.



Nicotinic acetylcholine receptors (nAChRs) belong to the ligand-gated ion channel superfamily [23,24]. There are a large number of nAChR subunits, such as *α*1-10, *β*1-4, *γ*, *δ*, and *ε*, which may mediate analgesic effects and adverse reactions [25,26]. Targeting specific nAChRs subtypes may reduce adverse reactions while maintaining efficient analgesia and becoming a real new analgesic target. All new compounds (**1**–**6**) were evaluated for their activity at ACh-evoked currents mediated by human (h) *α*1*β*1*εδ*, *α*1*β*1*γδ*, *α*3*β*2, *α*3*β*4, *α*4*β*2, *α*7, and *α*9*α*10 nAChRs, among which compound **2** showed significant inhibitory activity at all subtypes (>70% inhibition) except *α7* (Figure 3, Table S1). Interestingly, compound **2** selectively inhibited (>98% inhibition, n = 8–11) the *α*1*β*1*εδ* and *α*1*β*1*γδ* subtypes. For comparison, the α-conotoxin GI peptide from the marine cone snail *Conus geographus* venom antagonizes ACh-evoked currents mediated by h*α*1*β*1*εδ* with a half-maximal inhibitory concentration (IC50) of 20 nM [27]. According to the structural features of **1**–**6**, the preliminary structure–activity relationship (SAR) of inhibitory activities was tentatively discussed. Generally, compounds **1–3** exhibited stronger activities than compounds **4**–**6**, indicating that the presence of tetrahydropyridoindoles unit decreases inhibitory activities. Psegynamide B (**2**) showed stronger activities than psegynamide A (**1**), suggesting that C-terminal replacement with the methoxy group was beneficial to the activity.

**Figure 3.** Bar graph of compounds (**1**–**6**) (100 *μ*M) inhibition of ACh-evoked peak current amplitude mediated by human (h) *α*1*β*1ε*δ, α*1*β*1*γδ*, *α*3*β*2, *α*3*β*4, *α*4*β*2, *α*7 and *α*9*α*10 nAChRs. Whole-cell currents at h*α*1*β*1*εδ* and h*α*1*β*1*γδ* were activated by 5 *μ*M ACh, h*α*3*β*2 and h*α*9*α*10 were activated by 6 *μ*M ACh, and h*α*3*β*4, h*α*4*β*2, and h*α*7 were activated by 300, 3, and 100 *μ*M ACh, respectively (mean ± SD, n = 6–11).

To explain the different inhibitory activities and obtain further insight into the mechanism of nAChRs inhibition, molecular docking studies were carried out to explore the possible binding modes of compounds **1–6** and key interactions with nAChRs. The crystal structure of h*α*4*β*2 (PDB code: 5KXI) was used for further docking. The results in the docking study matched well with the inhibition activities (Table S2), compounds with lower calculated docking scores are considered to have higher binding affinities with the target. Among them, compound **2** showed the highest binding affinity to h*α*4*β*2 with the most negative free binding energy (−4.2 kcal/mol). Analysis for optimized binding conformation of compound **2** displayed that the hydrogen at N-6 in Trp interacts with Ser A180 through a hydrogen bond with a distance of 2.2 Å, and the Trp formed H-pi stacked bonds with Gln B50 with a distance of 4.0 Å (Figure 4). While the Trp residue was replaced by the tetrahydropyridoindoles unit, the H-H and H-pi bond was lost. In addition, the NH in Val (2) of compound **2** interacts with Asp A49 through a hydrogen bond with a distance of 2.5 Å. It is interesting to find that the Arg B45 may be important for the activity because of that NH2 in Trp and the C=O in Phe-OCH3 of compound **2** simultaneously interact

with Arg B45 through a hydrogen bond with distance of 2.2 Å and 3.3 Å, respectively. Furthermore, considering 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 [28,29]. The above findings provided that compound **2** might be helpful in developing different analgesic drugs by inhibiting nicotinic acetylcholine receptors.

**Figure 4.** Docking models of compound **2** with the crystal structure of h*α*4*β*2 (PDB code: 5kxi). (**A**) H-bonding interactions of compound **2** with h*α*4*β*2. (**B**) 2D schematic diagram of compound **2** with h*α*4*β*2. The H-bonds and H-pi stacked bonds are shown in yellow dashed lines and blue dashed lines, respectively.
