Effect of lathyrane-type diterpenoids in neural stem cell physiology: Microbial transformations, molecular docking and dynamics studies

Effect of lathyrane-type diterpenoids in neural stem cell physiology: Microbial transformations, molecular docking and dynamics studies Felipe Escobar-Monta˜ no a , Ricardo G´ omez-Oliva b,c , Abdellah Ezzanad d , Sonia V´ azquez de G´ orgolas b , David Zorrilla e,f , Antonio J. Macías-S´ anchez a,d , Jos´ e M. Botubol-Ares a,g,* , Pedro Nunez-Abades h , Carmen Castro b,c , Rosa Dur´ an-Patr´ on a,d,* , Rosario Hern´ andez-Gal´ an a,d a Departamento de Química Org´ anica, Facultad de Ciencias, Universidad de C´ adiz, Puerto Real C´ adiz, Spain b ´ Area de Fisiología, Facultad de Medicina, Universidad de C´ adiz, C´ adiz, Spain c Instituto de Investigaci´ on e Innovaci´ on Biom´ edica de C´ adiz (INiBICA), C´ adiz, Spain d Instituto Universitario de Investigaci´ on en Biomol´ eculas, Universidad de C´ adiz, Puerto Real C´ adiz, Spain e Departamento de Química Física, Facultad de Ciencias, Universidad de C´ adiz, Puerto Real C´ adiz, Spain f Instituto Universitario de Microscopía Electr´ onica y Materiales, Universidad de C´ adiz, Puerto Real C´ adiz, Spain g Instituto Universitario de Investigaci´ on Vitivinícola y Agroalimentaria, Universidad de C´ adiz, Puerto Real C´ adiz, Spain h Departamento de Fisiología, Universidad de Sevilla, Sevilla, Spain ARTICLE INFO Keywords: Mucor circinelloides Lathyrane Biotransformation NSC TGF α NRG1 Molecular docking Molecular dynamics ABSTRACT Promoting endogenous neurogenesis for brain repair is emerging as a promising strategy to mitigate the functional impairments associated with various neurological disorders characterized by neuronal death. Diterpenes featuring tigliane, ingenane, jatrophane and lathyrane skeletons, frequently found in Euphorbia plant species, are known protein kinase C (PKC) activators and exhibit a wide variety of pharmacological properties, including the stimulation of neurogenesis. Microbial transformation of these diterpenes represents a green and sustainable methodology that offers a hitherto little explored approach to obtaining novel derivatives and exploring structure–activity relationships. In the present study, we report the biotransformation of euphoboetirane A (4) and epoxyboetirane A (5), two lathyrane diterpenoids isolated from Euphorbia boetica, by Mucor circinelloides MC NRRL3631. Our findings revealed the production of nine biotransformation products (6–14), including jatrophane derivatives originated through an unprecedented rearrangement from the parent lathyranes. The chemical structures and absolute configurations of the new compounds were elucidated through comprehensive analysis using NMR and ECD spectroscopy, as well as MS. The study evaluated how principal metabolites and their derivatives affect TGF α and NRG1 release, as well as their potential to promote proliferation or differentiation in cultures of NSC isolated from the SVZ of adult mice. In order to shed some light on the mechanisms underlying the ability of 12 as a neurogenic compound, the interactions of selected compounds with PKC δ-C1B were analyzed through molecular docking and molecular dynamics. Based on these, it clearly appears that the ability of compound 12 to form both acceptor and donor hydrogen bonds with certain amino acid residues in the enzyme pocket leads to a higher affinity compound-PKC complex, which correlates with the observed biological activity. 1. Introduction Nature provides a continuous source of low molecular weight compounds of relevance in disease treatments. They have properties such as intrinsic cellular permeability, chemical diversity and specificity to molecular targets which originate from the evolutive pressure on organisms and are absent in compounds from combinatorial chemistry libraries [1,2]. The genus Euphorbia is distinguished by a high diversity of chemical constituents, encompassing structurally related macrocyclic and * Corresponding authors. E-mail addresses: [email protected] (J.M. Botubol-Ares), [email protected] (R. Dur´ an-Patr´ on). Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg https://doi.org/10.1016/j.bioorg.2024.107769 Received 28 June 2024; Received in revised form 26 August 2024; Accepted 27 August 2024 Bioorganic Chemistry 153 (2024) 107769 Available online 30 August 2024 0045-2068/© 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). polycyclic diterpenoid polyesters, including jatrophanes, tiglianes, ingenanes, and lathyranes [3–8]. Lathyrane-type diterpenoids feature a 5/11/3-tricyclic ring system that is frequently highly oxygenated and acylated. These compounds have been the subject of considerable interest due to their relevant biological activities [9], including modulation of multidrug resistance (MDR) [10], cytotoxicity against tumor cell lines [11], antiinflammatory properties [12], protein kinase C (PKC) mediated anti- HIV [13–15], and brain repair in response to neuronal death [16,17]. Brain repair is a currently a critical area of neuroscience focused on restoring brain tissue and function lost due to injuries of different types [18]. Stimulating endogenous neurogenesis, the process by which new neurons are generated from neural stem cells (NSCs) within the brain, is of paramount importance in this field [19]. Unlike other tissues, the brain has a limited capacity for self-repair, making the search for pharmacological agents that promote neurogenesis a promising therapeutic strategy [20,21]. Enhancing the brain’s natural ability to generate new neurons can help replace those lost to conditions such as stroke [22], traumatic brain injury [16,21,23], and neurodegenerative diseases like Alzheimer’s [24,25] or even infectious [26]. By fostering the growth and integration of new neurons, therapies aimed at stimulating endogenous neurogenesis hold the potential to improve cognitive function, restore motor skills, and ultimately enhance the quality of life for individuals affected by neurological disorders [25]. However, the search for pharmacological tools requires the identification of targeting molecules and the design of agents that modulate the activity of such targets. This is the case of PKC isozymes. Their capacity to selectively promote the release of different growth factors makes them crucial targets to promote endogenous neurogenesis [27]. In previous reports, we have described ingol-type lathyranes with the ability to promote neurogenesis by stimulating the expansion (ELAC, 1) [17] of subventricular zone (SVZ)-derived NSCs via the modulation of different PKC isozymes (Fig. 1) as well as their differentiation into functional neurons (EOF2, 2) [16]. ELAC (1) induced SVZ-derived NSCs proliferation in vitro and stimulated neurogenesis in both the SVZ and the dentate gyrus (DG) of the hippocampus in vivo in a murine model. However, its C-7 acetylated derivative (1a) did not induce any effect on the in vitro proliferation assay [17]. Hence, it can be inferred that a free hydroxyl group at the C-7 position is crucial for the compound’s binding to the C-1 regulatory domain of PKC, in a manner analogous to that observed with the C-20 position of phorbol esters [28]. In contrast, EOF2 (2) promoted the differentiation and survival of neuroblasts in cultures of NSCs isolated from the SVZ [16], while exhibiting no effect on proliferation assays. It is noteworthy that this compound demonstrated remarkable efficacy in differentiating neuroblasts in vivo in a murine model of mechanical cortical brain injuries, promoting the migration of newly generated neuroblasts from the SVZ towards the injury site [16]. In contrast, the diterpene EOF3 (3) a structurally similar molecule, did not exhibit any effect on SVZ cell proliferation or differentiation [16]. Moreover, euphoboetirane A (4) and epoxyboetirane A (5), two lathyranes previously isolated from Euphorbia boetica, exhibited a notablecapacity to promote NSCs proliferation [29]. Despite the accumulated evidences on the role of lathyrane-type diterpenes on NSC physiology, there is still limited knowledge about the molecular mechanisms underlying the differential effect that the above-mentioned diterpenes exert on NSC. Recent evidence suggests that the activation of classical PKC by diterpenes promotes the secretion of neurotrophic factors that bind to the epidermal growth factor receptor (EGFR) [30] and induce the NSCs proliferation [31]. This has been observed with diterpenes that facilitate the release of the transforming growth factor alpha (TGF α ) [25,32]. Similarly, the activation of novel PKC isozymes promotes the release of neuregulin 1 (NRG1), a growth factor involved in the differentiation of NSCs into a neuronal phenotype [16]. Thus, the capacity of lathyranes to induce TGF α release may be an indirect measure to predict their role as proliferative agents [28]. Similarly, testing the capacity of a particular lathyrane to promote NRG1 release may give an indirect idea of their potential as differentiating agents. Consequently, these assays may be useful in structure–activity relationship studies shedding some light on the mechanism of action of these lathyrane derivatives. Furthermore, these studies would facilitate the identification of compounds useful as pharmacological agents for the treatment of disorders in which neurogenesis is altered. Biocatalysis, a green and sustainable methodology [33], represents a promising avenue for enhancing the structural diversity of lathyranes [34–36]. Although diterpenes exhibit good tolerance towards microbial transformations, there have been limited studies exploring the biotranformation of lathyrane-type diterpenoids. Previous studies have revealed that lathyrol derivatives can be modified by several microorganisms, leading to regioselective hydroxylations, glycosylations, deoxygenations, and cyclopropane ring opening [37–39]. Furthermore, we recently reported the biotransformation of euphoboetirane A (4) by Streptomyces puniceus BC-5 GB.11, which led to an unusual isomerization of the Δ 12 double bond and a cyclopropane rearrangement, together with regioselective oxidation at non-activated carbons [40]. Therefore, as a part of our ongoing research on bioactive compounds for the treatment of neurodegenerative disorders associated with cognitive impairment, this study describes the biotransformation of Fig. 1. Lathyrane-type diterpenes previously tested on NSCs proliferation or differentiation. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 2 euphoboetirane A (4) and epoxyboetirane A (5), the major lathyranetype diterpenoids found in E. boetica, by Mucor circinelloides MC NRRL3631. Furthermore, the capacity of the major biotransformation compounds and their p-methoxyphenylacetyl derivatives to promote the TGF α and NRG1 release, as well as their subsequent role in NSC physiology, was evaluated. Finally, the interaction between selected compounds and PKC δ-C1B was analyzed using molecular docking and molecular dynamics to identify the putative binding mode of these compounds to the enzymatic pocket. 2. Results and discussion The fresh aerial parts of E. boetica were ground into a fine powder and subsequently extracted with methanol. The n-hexane-soluble fraction was then subjected to column chromatography to obtain euphoboetirane A (4) and epoxyboetirane A (5). The structures of these compounds were elucidated by comparing their spectroscopic data with the values reported in the literature [41,42]. 2.1. Biotransformation of lathyranes 4 and 5 by Mucor circinelloides NRRL3631 Eleven microorganisms were screened for their ability to biotransform euphoboetirane A (4). Among them, only M. circinelloides NRRL3631 was found to biotransform this lathyrane derivative. The preparative-scale microbial transformation of this substrate furnished four new hydroxylated lathyranes: 6 (51.6 % yield), 7 (0.6 % yield), 8 (0.6 % yield), and 9 (0.4 % yield). Additionally, two new rearranged compounds with jatrophane skeleton, 10 (3.4 % yield) and 11 (3.8 % yield), were obtained (Scheme 1A). Compound 6 was isolated as an amorphous solid and exhibited a sodiated molecular ion at m/z 499.2307 ([M +Na] + ), indicative of the molecular formula C 26 H 36 O 8 Na, as determined through HRESIMS analysis. Compared to 4 [41], its 13 C NMR spectra (Fig. S2) showed a new oxygenated methine carbon signal at δ C 66.7, which correlated to the signal at δ H 4.12–4.08 (m) in the gHSQC experiment (Fig. S4). This methine group was assigned to C-8 based on the long range HMBC correlations from H-8 (δ H 4.12–4.08, m) to C-6 (δ C 141.4), C-7 (δ C 45.1), C-9 (δ C 40.1), and C-10 (δ C 24.9) (Tables 1 and 2), as well as the 1 H– 1 H COSY correlations of H 2 -7/H-8/H-9. Furthermore, the key 1D and 2D NOESY correlations for H-5/H-12/H-8/H 3 -19 suggested the β-orienta- tion of these protons, while correlations for H-9/H-11/H 3 -18 supported their α -orientation (Figs. S6 and S7). Therefore, the NOESY correlations pointed to an α -orientation of the hydroxyl group at C-8, in contrast to that described by Wu et al. for the regioselective hydroxylation at C-8 of Euphorbia factor L 3 by M. circinelloides [37]. The absolute configuration of the carbon C-8 was deduced using the Mosher NMR method. This involved treating compound 6 separately with (R)-(−)- α -methoxyphenylacetic acid (R-(−)-MPA) and (S)- (+)- α -methoxyphenylacetic acid (S-(+)-MPA), as outlined in the experimental section, resulting in the formation of the corresponding (R)- and (S)-MPA esters of 6, respectively [43]. A comparison of the 1 H NMR spectra (Fig. S10 and S11) of the two esters showed a positive value of Δδ RS for the vicinal proton H-7β (+0.24 ppm) and a negative value of Δδ RS for H-9 (−0.24 ppm). This indicated an 8R configuration in compound 6. Consequently, the structure for compound 6 was proposed as (8R)-8-hydroxyeuphoboetirane A, which suggests that the configuration proposed by Wu et al. should be revised [37]. Compound 7 showed a molecular formula C 26 H 36 O 8 , as determined by HRESIMS (m/z 499.2312 [M +Na] + ) (Fig. S18), which matched that of compound 6. The spin systems inferred from 1 H– 1 H COSY correlations (Fig. S14) displayed identical connectivity to that of compound 6, suggesting that it might be an isomeric compound. Its 1 H NMR spectrum (Fig. S12) showed as main difference the deshielding for H-8 (δ H 4.82–4.77 in 7 vs δ H 4.12–4.08 in 6) and H-12 (δ H 7.19 in 7 vs δ H 6.42 in 6) (Table 1). These features indicated a distinct configuration at C-8 in compound 7. 2D NOESY (Fig. S17) correlations for H-7 α /H-8/H-9, H-9/ H 3 -18 and H-4/H 2 -17/H-7 α suggested that these protons are α -oriented, while the correlations for H-12/H 3 -19/H-5/H-7β were used to place these protons in the opposite face of the molecule. Based on these correlations, the configuration at C-8 was established as epimeric to that of 6. Accordingly, the structure of 7 was identified as (8S)-8-hydrox- yeuphoboetirane A. The HRESIMS of compound 8 gave a sodium adduct ion peak at m/z 499.2313 [M +Na] + , consistent with the formula C 26 H 36 O 8 Na, leading to the molecular formula C 26 H 36 O 8 . Additionally, the presence of an oxygenated methylene carbon signal at δ C 63.8, attached to a multiplet signal at δ H 3.77–3.68 in the gHSQC experiment (Fig. S23), pointed to a hydroxylation of the parent compound. The disappearance of one methyl signal of the gem-dimethylcyclopropyl ring in the 1 H NMR Scheme 1. A) Biotransformation of euphoboetirane A (4) by Mucor circinelloides NRRL3631 and chemical esterification of compound 6. B) Biotransformation of epoxyboetirane A (5) by M. circinelloides NRRL3631 and chemical esterification of compound 12. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 3 spectrum (Fig. S20), the HMBC cross-peaks from δ H 1.27 (H 3 -18), 1.49 (H-11) and 1.33–1.28 (H-9) to δ C 63.8 (C-19), and the 1D NOESY correlations for H 3 -18/H-11/H-9/H 3 -20 and H 2 -19/H-12/H 2 -8 located the hydroxyl group at C-19. Consequently, the structure for 8 was proposed as (10S)-19-hydroxyeuphoboetirane A. These results demonstrated a different regioselectivity of M. circinelloides compared to that previously described with S. puniceus [40]. While S. puniceus catalyzed hydroxylation of 4 at C-18, M. circinelloides targeted C-19 instead. Compound 9 possessed a molecular formula of C 26 H 36 O 8 , as deduced from the HRESIMS analysis (m/z 499.2303 [M +Na] + ) (Fig S34). This compound shared the same spin system as 6, suggesting it could be an isomer. A comparison of the 1 H NMR spectra of both compounds showed as the main difference the shielding of the signal corresponding to H-12 from δ H 6.42 in 6 to δ H 5.23 in 9 (Table 1), pointing to a configurational change at C-12. The configuration of the double bond can also influence the NMR chemical shift of the double bond carbons and the carbons directly joined to it [44,45]. Thus, the 13 C NMR spectrum of compound 9 (Fig. S29) showed a shift of the signals corresponding to C-12 and C-20 to upfield and downfield, respectively, compared to compound 6 (δ C 133.6 for C-12 and δ C 22.2 for C-20 in 9; δ C 144.8 for C-12 and δ C 12.3 for C-20 in 6) (Table 2). These data pointed to a (Z)-configuration for the C- 12/C-13 double bond. Careful analysis of 1D NOESY spectra (Fig.s S33a- g) indicated correlations for H-12/H 3 -20/H 3 -19/H-8 supporting the (Z)- configuration of the trisubstituted C-12/C-13 double bond and the α -orientation of the hydroxyl group at C-8. This particular configuration of the C-12/C-13 double bond has only been previously described for Euphorbia factor L 2a , which was obtained from the seeds of E. lathyris [46]. Recently, we reported a similar configuration for a biotransformation product of 4 by S. puniceus [40]. Further 1D NOESY correlations between H 3 -18/H-11/H 3 -19/H-9 supported the assignment of the absolute configuration for 9 as (8R,12Z)-8-hydroxyeuphoboetirane A. Compounds 10 and 11 showed sodium adduct ions at m/z 499.2313 and 499.2312, respectively, consistent with the formula C 26 H 36 O 8 Na as deduced from the HRESIMS analysis (Figs. S43 and S52). Both compounds showed spectroscopic features within the 5-membered ring similar to those of 4. Conversely, two isolated spin systems of H 2 -7/H-8/ H-9 and H-11/H-12 were identified in their 1 H– 1 H COSY spectra (Figs. S38 and S47), indicating a cleavage of the C-9/C-11 bond within the cyclopropane ring. Indeed, the characteristic proton and carbon resonances for the gem-dimethyl-substituted cyclopropane ring were absent. Furthermore, their NMR spectra (Figs. S36 and S45) showed olefinic protons located at C-8 and C-9, alongside a hydroxyl group located at C-11, as evidenced by HMBC correlations from H-11 (δ H 4.16 in 10, δ H 4.29 in 11) to C-10 (δ C 42.4 in 10, δ C 42.5 in 11), C-12 (δ H 142.6 in 10, δ H 139.1 in 11), C-13 (δ C 132.8 in 10, δ C 135.3 in 11), C-18 (δ C 17.8 in 11), and C-19 (δ C 26.4 in 11) (Tables 1 and 2). HMBC correlations from H 3 -18 and H 3 -19 to C-11 and from H-11, H 3 -18 and H 3 -19 to C-9 confirmed the cyclopropane rearrangement to a jatrophane skeleton. The relative stereochemistry of 10 and 11 was elucidated through the analysis of 1D and 2D NOESY spectra (Fig. 2) and the examination of coupling constants. The configuration of the double bond C-8/C-9 was assigned as E for both compounds based on the large coupling constant observed between H-8 and H-9 (J 8-9 =15.3 Hz in 10 and J 8-9 =15.2 Hz in 11). 1D and 2D NOESY correlations observed in 10 for H 3 -20/H-11/ H 3 -18/H-8, H-7 α /H-17b and H-3/H-4 suggested that these protons are α -oriented (directed inward in the jatrophane skeleton), and H 3 -19/H-9/ H-7β and H-5/H-12 are β-oriented. On the contrary, the 1D and 2D NOESY cross-peaks in 11 of H-1 α /H-12/H 3 -18/H-8/H-4/H-2 revealed that these protons were α -oriented while H 3 -20/H-11/H 3 -19/H-9, H-7β/ H-17b and H-17a/H-5 lie on the β face of the molecule. The absolute configuration for compound 11 was determined by comparing its experimental electronic circular dichroism (ECD) spectrum with a theoretically computed ECD spectrum obtained using time-dependent density functional theory (TDDFT) calculations [47] with the Gaussian 16 program [48]. The calculated ECD spectrum for 11 closely matched the experimental spectrum (Fig. S54). Consequently, the above data supported an absolute configuration for 11 as (2S,3S,4R,5R,11S,15R)- 3,5,15-triacetoxy-11-hydroxyjatropha-6(17),8E,12E-trien-14-one whereas the absolute stereochemistry for 10 at C-11 was assigned as R. Incubation of M. circinelloides NRRL3631 with 5 resulted in the formation of three novel products, compounds 12 (8.4 % yield), 13 (1.0 % yield), and 14 (0.6 % yield) (Scheme 1B). HRESIMS of compound 12 (Fig. S62) gave a sodium adduct ion peak at m/z 515.2256 [M +Na] + , which is consistent with the formula C 26 H 36 O 9 Na for the molecular ion. This indicated the presence of an additional oxygen atom in the structure of 12 compared to the parent compound 5. Moreover, the 1 H and 13 C NMR spectroscopic data of compound 12 (Table 3, Figs. S55-56) were similar to those of 6, suggesting a hydroxylation at C-8. This was supported by a signal at δ H 4.19, absent in compound 5, which correlated with the carbon at δ C 66.4 in the HSQC spectrum and connected to H 2 -7 and H-9/H-11/H-12 in the 1 H– 1 H COSY spectrum. 1D NOESY Table 1 1 H NMR Spectroscopic data for compounds 6–11 (400 MHz) in CDCl 3 (δ H in ppm, J in Hz). Position 6 7 8 9 10 11 1 α 3.42, dd (14.4, 8.9) 3.39, dd (14.0, 8.1) 3.42, dd (14.4, 8.7) 3.15, dd (13.3, 6.4) 3.05, dd (12.8, 7.5) 3.00, dd (14.4, 8.0) 1β1.60–1.51, m 1.60–1.53, m 1.61–1.55, m 1.70–1.64, m 1.45, dd (12.8, 11.5) 1.96–1.93, m 2 2.26–2.20, m 2.29–2.17, m 2.30–2.18, m 2.17–2.08, m 2.47–2.38, m 2.24–2.15, m 3 5.54, t (3.5) 5.51, t (3.5) 5.55, t (3.5) 5.52–5.49, m 5.42, t (6.5) 5.60–5.57, m 4 2.71, dd (10.2, 3.5) 2.70, dd (10.5, 3.5) 2.76, dd (10.3, 3.5) 2.59, dd (10.3, 4.1) 2.70, dd (6.5, 1.6) 2.78, m 5 6.03, d (10.2) 6.25, d (10.5) 6.06, d (10.3) 5.81, d (10.3) 6.06, br s 5.60–5.57, m 7 α 2.37, dd (13.2, 10.6) 2.39, br d (15.8) 2.30–2.18, m 2.40, dd (15.4, 9.2) 2.98, dd (14.4, 8.7) 2.88, dd (14.0, 4.9) 7β2.30, d (13.2) 2.55, dt (15.8, 7.4) 2.10–2.01, m 2.35–2.29, m 2.86, dd (14.4, 5.7) 2.82, dd (14.0, 9.0) 8 α −4.82–4.77, m 1.86–1.75, m 3.58, t (9.2) 5.21, ddd (15.3, 8.7, 5.7) 5.39–5.29, d (15.2, 9.0, 4.9) 8β4.12–4.08, m − − 9 1.25.1.22, m 1.26–1.22, m 1.33–1.28, m 0.92–0.88, m 5.74, d (15.3) 5.43, d (15.2) 11 1.60–1.51, m 1.60–1.53, m 1.49, dd (11.5, 8.1) 1.70–1.64, m 4.16, d (9.0) 4.29, d (9.2) 12 6.42, dd (11.6, 1.2) 7.19, dd (11.2, 1.0) 6.53, dd (11.5, 1.4) 5.23, dd (9.4, 1.6) 6.72, dd (9.0, 1.3) 6.02, dd (9.2, 1.4) 16 0.89, d (6.8) 0.90, d (6.7) 0.90, d (6.7) 0.91, d (6.6) 1.01, d (6.9) 0.96, d (6.7) 17a 5.00, s 5.03, s 4.98, br s 5.52–5.49, m 5.12, s 4.99, s 17b 4.72, s 4.87, s 4.73, br s 5.05, br s 5.06, s 4.91, s 18 1.21, s 1.19, s 1.27, s 1.11, s 0.94, s 1.03, s 19 1.28, s 1.39, s 3.77–3.68, m 1.25, s 1.18, s 1.16, s 20 1.64, d (1.2) 1.69, d (1.0) 1.69, d (1.4) 1.76, d (1.6) 1.75, d (1.3) 1.90, d (1.4) OCOCH 3 -3 2.02, s 2.01, s 2.03, s 2.02, a s 2.03, s 2.08, c s OCOCH 3 -5 1.98, s 1.98, s 1.97, s 1.97, s 1.98, b s 2.06, c s OCOCH 3 -15 2.08, s 2.09, s 2.14, s 2.18, a s 2.12, b s 2.07, s a-c Interchangeable signals. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 4 correlations observed for H 3 -19/H-8/H-12/H-5/H-7β indicated that these protons are β-oriented. Conversely, the presence of correlations between H-9/H-11/H 3 -18/H 3 -20 supported and α -orientation, allowing the assignment of an α -stereochemistry for the hydroxyl group at C-8. These correlations allowed us to assign the structure of compound 12 as (8R)-8-hydroxyepoxyboetirane A. The molecular formula of compounds 13 and 14 was established as C 26 H 36 O 9 based on the pseudo-molecular ions observed at m/z 515.2252 and 515.2266 ([M +Na] + ), respectively, in positive HRESIMS. Their NMR data (Table 3) showed that 13 and 14 were highly similar to 10 and 11, respectively, suggesting that both compounds could have a jatrophane skeleton. HMBC correlations for 13 and 14 from H 3 -18 and H 3 -19 to C-11 and from H-11, H 3 -18 and H 3 -19 to C-9 corroborated a cyclopropane ring opening. Moreover, the presence of a methine a δ C 74.8 in 13 and 75.0 in 14, which correlated with a signal at δ H 4.14 and 4.31, respectively, in the HSQC experiments (Figs. S67 and S75) and with H-12 in their 1 H– 1 H COSY spectra (Figs. S66 and S74), pointed to a hydroxylation at C-11. 2D NOESY correlations for 13 were similar to those observed for compound 11. C-11 Hydroxyl group in 13 was assigned β based on the 2D NOESY correlations between H-11/H 3 -18/ H 3 -20 (oriented inward in the jatrophane skeleton) and the correlations between H 3 -19/H-12/H-5/H-17a on the opposite side of the macrocycle (Fig. S69). On the other hand, 2D NOESY correlations for 14 between H 3 -18/H-12/H-4/H-2 revealed their co-facial relationship, which suggests an α -orientation (Fig. S77). Additionally, the 1D NOESY crosspeaks between H 3 -20/H-11/H 3 -19 demonstrated that these protons were also co-facial, thereby supporting a β-orientation (Fig. S78). Based on these findings, the structures of 13 and 14 were identified as (2S,3S,4R,5R,6S,11R,15R)-3,5,15-triacetoxy-6(17)-epoxy-11-hydrox- yjatropha-8E,12E-dien-14-one and (2S,3S,4R,5R,6S,11S,15R)-3,5,15- triacetoxy-6(17)-epoxy-11-hydroxyjatropha-8E,11E-dien-14-one, respectively. 2.2. Effect of lathyrane-type diterpenes on the PKC-dependent release of TGF α and NRG1 As previously stated, lathyrane-type diterpenes represent a novel class of drug candidates that have been shown to modulate adult neurogenesis through the PKC activation. These small molecules exert their effects by activating specific PKC isozymes, which phosphorylate ligands of the ErbB family of receptors and act as growth factors involved in either proliferation (such as TGF α ) or differentiation (such as NRG1). These growth factors are synthesized as transmembrane proteins, in which the active ligand is located in their extracellular N-terminal domains and need to be proteolytically processed to release the biologically active mature ligand. This release is catalyzed by the metalloprotease ADAM17, which selects its substrates depending on PKC-dependent phosphorylation reactions that act on the growth factor to be cleaved [49,50]. The lathyranes euphoboetirane A (4), epoxyboetirane A (5), and their C-8 hydroxylated biotransformation derivatives (6 and 12) were evaluated for their ability to stimulate TGF α and/or NRG1 release. Additionally, given the potential significance of the p-methox- yphenylacetoxy chain in EOF2 (2) on NSC differentiation, the impact of this chain on the activity of the tested compounds was investigated. Consequently, derivatives 15 and 16, containing the p-methox- yphenylacetate group at the C-8 position, were synthesized and evaluated. To assess the activity of these compounds, we built cDNA constructs encoding fusion proteins in which TGF α or NRG1 were flanked by the green fluorescent protein EGFP on their C-terminal cytoplasmic domain and by the red fluorescent protein mCherry on its N-terminal extracellular domain (Figs. 3A and 4A). The cDNA was transfected into HEK293 cells. As reported before, this cell line expresses ADAM17, as well as classical and novel PKC isozymes making it a suitable model to study the ADAM17 dependent release of TGF α or NRG1 stimulated by our compounds [16]. Once cells expressed the fusion protein, using time-lapse real-time confocal microscopy studies, we analyze the release of either TGF α or NRG1 in the presence and absence of the potential PKC activating compounds. As shown in Figs. 3B and 4B, the expression of the pro-TGF α and pro-NRG1 ligands inserted in the plasma membrane can be observed by the fluorescent signal that results of the combination of mCherry and EGFP (with a 1/1 ratio mCherry/EGFP). As the shedding of the red fluorescent (mCherry-linked) soluble ligand occurs, the mCherry/EGFP fluorescence ratio is reduced. In Fig. 3C-G the ratiometric images show that in cells expressing TGF α , the mCherry/EGFP fluorescence ratio does not change over the time course of 180 min in negative control cells in the absence of compounds (see supplementary movie 1). The addition of compounds 4, 15, and 16 at a final concentration of 1 µM did not significantly alter this ratio. A small, not very significant, reduction in the mCherry/EGFP ratio was observed when compounds 5, 6 and 12 (see supplementary movie 2) were added to the culture medium. However, this reduction did not reach a reduction comparable to the Table 2 13 C NMR Spectroscopic data for compounds 6–11 (100 MHz) in CDCl 3 (δ C in ppm). Position 6, type 7, type 8, type 9, type 10, type 11, type 1 48.1, CH 2 48.3, CH 2 48.4, CH 2 46.5, CH 2 44.2, CH 2 44.1, CH 2 2 37.0, CH 37.6, CH 37.2, CH 37.6, CH 35.8, CH 37.0, CH 3 80.1, CH 80.2, CH 80.2, CH 79.0, CH 75.2, CH 76.2, CH 4 52.0, CH 53.9, CH 52.2, CH 52.0, CH 50.4, CH 53.6, CH 5 66.3, CH 70.1, CH 65.7, CH 73.4, CH 68.8, CH 71.0, CH 6 141.4, C 143.9, C 144.2, a C 140.0, C 145.1, C 144.9, C 7 45.1, CH 2 42.4, CH 2 35.1, CH 2 38.3, CH 2 41.3, CH 2 40.8, CH 2 8 66.7, CH 70.1, CH 21.2, b CH 2 70.7, CH 125.7, CH 125.4, CH 9 40.1, CH 37.4, CH 35.4, CH 41.3, CH 138.8, CH 139.1, CH 10 24.9, C 26.1, C 31.7, C 21.7, C 42.4, C 42.5, C 11 29.7, CH 30.7, CH 27.9, CH 25.0, CH 75.9, CH 74.7, CH 12 144.8, CH 146.9, CH 144.3, a CH 133.6, CH 142.6, CH 139.1, CH 13 135.0, C 133.6, C 134.9, C 138.4, C 132.8, C 135.3, C 14 196.5, C 197.0, C 197.0, C 205.1, C 199.2, C 199.0, C 15 92.4, C 91.9, C 92.3, C 91.9, C 90.3, C 91.2, C 16 14.1, CH 3 13.9, CH 3 14.1, CH 3 13.3, CH 3 14.0, CH 3 13.9, CH 3 17 116.4, CH 2 117.2, CH 2 115.7, CH 2 117.3, CH 2 114.5, CH 2 115.5, CH 2 18 28.8, CH 3 30.9, CH 3 24.3, CH 3 28.3, CH 3 23.6, CH 3 17.8, CH 3 19 16.3, CH 3 17.2, CH 3 63.8, CH 2 15.7, CH 3 23.0, CH 3 26.4, CH 3 20 12.3, CH 3 12.4, CH 3 12.5, CH 3 22.2, CH 3 13.1, CH 3 14.5, CH 3 OCO-3 170.6, C 170.6, C 170.7, C 170.3, C 170.6, C 170.4, f C OCOCH 3 - 3 20.8, CH 3 20.8, CH 3 20.9, b CH 3 20.8, c CH 3 21.0, CH 3 21.0, g CH 3 OCO-5 170.8, C 172.3, C 170.6, C 169.4, C 169.7, d C 169.5, f C OCOCH 3 - 5 21.2, CH 3 21.3, CH 3 21.2, b CH 3 21.0, CH 3 21.4, e CH 3 21.5, g CH 3 OCO-15 169.7, C 169.9, C 170.0, C 170.3, C 170.0, d C 170.2, C OCOCH 3 - 15 21.9, CH 3 21.9, CH 3 21.6, b CH 3 21.9, c CH 3 21.3, e CH 3 21.2, g CH 3 a-g Interchangeable signals. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 5 positive control cells stimulated with compound ER272 (supplementary movie 3), which was reported previously as a classical PKC activating compound that stimulates the release of TGF α [32]. These results suggested that compounds 4, 15 and 16 were not stimulating the release of TGF α probably because they were not activating classical PKC. On the contrary, compounds 5, 6 and 12 were able to marginally stimulate the release of TGF α , which may suggest its low potency at activating classical PKC. Previous reports show that compounds that activate classical PKC isozymes and stimulate the release of the EGF receptor (EGFR) ligand TGF α promote the proliferation of NSC and undifferentiated progenitors in vitro and in vivo in adult mouse models [28,32,51]. Thus, in order to understand whether these compounds were able to promote proliferation of NSC despite their low capacity to induce TGF α release, we then analyzed the capacity of these compounds to stimulate proliferation in neurosphere cultures of cells isolated from the SVZ of 7-day postnatal mice. NSC were cultured under floating conditions for 72 h in the presence and absence of the compounds until they formed neurospheres. The area and the number of the neurospheres formed was then analyzed as an indirect measure of proliferation. Control cultures were grown in the presence of bFGF as growth factor. As shown in Fig. S89, the area of the neurospheres in positive control cultures grown in the presence of the EGFR ligand, showed a nearly 10-fold increase in the neurosphere area, whereas the use of compounds 4–6, 12, 15, and 16 did not show any effect on neurosphere area. These results were useful to understand those observed in the TGF α release assay and indicated that the small concentrations of TGF α released in response to compounds 5, 6 and 12 were not enough to promote proliferation. Fig. 2. Selected 1D and 2D NOESY correlations for 10 and 11. Correlations on the β-face are highlighted in red. Correlations on the α -face correlations are highlighted in blue. Correlations involving nuclei on the α -face and β-face are indicated in black. Table 3 1 H and 13 C NMR Spectroscopic data for compounds 12–14 in CDCl 3 (δ H in ppm, J in Hz). Position 12 13 14 δ Ha δ C, b type δ Ha δ C , b type δ Ha δ C , b type 1 α 3.38, dd (14.3, 8.3) 47.6, CH 2 3.08, dd (13.3, 7.9) 44.3, CH 2 3.07, dd (14.5, 8.2) 43.8, CH 2 1β1.51, dd (14.3, 11.9) 1.37, dd (13.3, 11.1) 1.87–1.79, m 2 2.14–2.05, m 37.5, CH 2.44–2.37, m 35.8, CH 2.34–2.25, m 37.1, CH 3 5.46, t (3.4) 80.1, CH 5.31, t (6.5) 75.3, CH 5.54–5.50, m 76.1, CH 4 1.87, dd (8.9, 3.4) 49.8, CH 2.51–2.49, m 49.0, CH 2.94–2.89, m 51.3, CH 5 6.10, d (8.9) 66.0, CH 6.19, d (2.5) 65.7, CH 5.54–5.50, m 67.3, CH 6−57.5, C −59.6, C −58.2, C 7 α 1.26–1.23, m 43.3, CH 2 2.29, dd (14.6, 3.6) 38.2, CH 2 2.46–2.39, m 38.0, CH 2 7β2.19, dt (13.8, 2.3) 2.24–2.19, m 2.15–2.09, m 8 4.19, td (10.3, 2.3) 66.4, CH 5.56–5.48, m 122.7, CH 5.54–5.50, m 122.3, CH 9 1.17, dd (10.3, 8.6) 39.6, CH 5.56–5.48, m 140.8, CH 5.54–5.50, m 140.7, CH 10 −25.3, C −42.2, C −42.1, C 11 1.67, dd (11.5, 8.6) 29.9, CH 4.14, dd (8.7, 5.8) 74.8, CH 4.31, dd (8.0, 5.0) 75.0, CH 11-OH − − 1.74, d (5.8) −1.68, d (5.0) − 12 6.54, dd (11.5, 1.4) 141.9, CH 6.74, dd (8.7, 1.4) 141.4, CH 6.10, d (8.0) 139.0, CH 13 −136.6, C −134.8, C −136.5, C 14 −196.6, C −199.0, C −198.7, C 15 −91.9, C −90.2, C −91.5, C 16 0.86, d (6.7) 13.9, CH 3 0.92, d (6.9) 13.9, CH 3 0.95, d (6.7) 13.9, CH 3 17a 2.49, d (3.4) 55.2, CH 2 2.80, d (4.6) 50.6, CH 2 2.73, d (4.8) 50.8, CH 2 17b 2.30, m 2.51, d (4.6) 2.48, d (4.8) 18 1.25, s 28.8, CH 3 1.07, s 25.2, CH 3 1.10, s 19.4, CH 3 19 1.34, s 16.2, CH 3 1.20, s 20.0, CH 3 1.19, s 25.6, CH 3 20 1.83, d (1.4) 12.2, CH 3 1.83, d (1.4) 13.1, CH 3 1.97, s 14.3, CH 3 OCO-3 −170.2, C −169.9, e C−170.2, h C OCOCH 3 -3 2.01, s 20.8, CH 3 2.07, d s 21.3, f CH 3 2.07, g s 21.5, i CH 3 OCO-5 −170.9, C −169.5, C −170.1, h C OCOCH 3 -5 2.09, s 21.8, c CH 3 2.10, s 21.2, CH 3 2.03, g s 21.0, i CH 3 OCO-15 169.6, C −170.0, e C−169.2, C OCOCH 3 -15 2.09, s 21.0, c CH 3 2.02, d s 21.0, f CH 3 2.05, s 21.0, i CH 3 a Recorded at 400 MHz; b Recorded at 100 MHz; c-i Interchangeable signals. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 6 Fig. 3. Effect of lathyrane-type diterpenes on the PKC-dependent release of TGF α . A. Schematic drawing representing the mCherry-TGF α -eGFP construct. B. Schematic representation of the mechanisms involved in the TGF α -bound fluorescence release. C. Quantitative analysis of the microscopic ratiometric images obtained from the time-lapse assays of HEK293T cells expressing mCherry-TGF α -eGFP and stimulated with 1 µM concentrations of 4–6, 12, 15, and 16. mCherry/eGFP ratios were normalized to the average mCherry/eGFP ratio measured before stimulation (see supplementary movies 1, 2 and 3 for representative time lapse recordings)). The mean normalized mCherry/eGFP ratios and SEM are shown, n =40. D-G. mCherry/eGFP ratiometric images at the indicated time points are shown in the intensity-modulated display mode. The color range goes from red to blue to represent mCherry/eGFP ratio. The upper and lower limits of the ratio range are shown on the bottom. Scale bar represents 25 μ m. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 7 Based on previous findings we hypothesized about the possibility that these compounds were activating novel PKC isozymes and therefore stimulated the release of NRG1 like lathyrane EOF2 (2) [16]. Thus, using an identical approach, as shown in Fig. 4, we tested the capacity of compounds 12, 15 and 16 to stimulate the release of NRG1. HEK293 cells were transfected with the eGFP-NRG1-mCherry (Fig. 4A-B). As shown Fig. 4C-G, no changes on the eGFP/mCherry fluorescence ratio were observed in cells transfected with the eGFP-NRG1-mCherry that had not been treated (see supplementary movie 4). However, the addition of compound 12 reduced this ratio (see supplementary movie 5) as much as the treatment with the positive control (EOF2 (2)) (see supplementary movie 6), indicating that compound 12 facilitated the shedding of the NRG1 ligand to the extracellular medium. On the contrary, neither compound 15 or 16 stimulated the release of NRG1. In the context of previous reports, which showed that compounds like EOF2 (2) activated novel PKC and stimulated the release of NRG1, these results suggest that 12 might be a novel PKC activating compound [16,52]. Interestingly, it has previously been shown that compounds that activate novel PKC promote differentiation of NSC cells towards a neuronal phenotype [16]. Thus, in order to analyze whether compound 12 was able to stimulate differentiation of NSC in vitro, NSC isolated from the SVZ were grown attached onto a substrate and were allowed to differentiate in the absence of growth factors and stimulated by compound 12. As shown in Fig. 5A-D, the addition of compound 12 at concentrations of up to 1 µM and 5 µM increased the percentage of beta- III-tubulin + cells, indicating that compound 12 induced the differentiation of NSC towards a neuronal phenotype (beta-III-tubulin + cells) in cultures of cells isolated from the mouse SVZ. However, compound 12 did not show any effect on the percentage of GFAP + glial cells (Fig. 5A-C, E), indicating that it did not have any effect on the differentiation of these cells towards a glial phenotype at any of the concentrations used. These results agreed with previous reports, which show that novel PKC activating compounds promote NRG1 release as well as differentiation of NSC in vitro and in vivo in a murine model of mechanical cortical brain injuries [16]. Existing therapeutics to promote neurogenesis include a variety of approaches such as pharmacological agents, lifestyle interventions, and advanced biological techniques. Pharmacologically, antidepressants like selective serotonin reuptake inhibitors (SSRIs) have been shown to enhance neurogenesis, particularly in the hippocampus [53]. Additionally, certain cognitive enhancers and neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), play a crucial role in supporting the growth and survival of new neurons [54]. Lifestyle interventions, including regular physical exercise and cognitive stimulation, have also been demonstrated to promote neurogenesis [55] also via the promotion of BDNF release. Advanced biological approaches, such as stem cell therapy and gene editing technologies like CRISPR, offer potential for directly enhancing the brain’s regenerative capabilities [56]. These diverse strategies highlight the multifaceted nature of promoting neurogenesis as a means to improve outcomes for individuals with neurological conditions. In here we are showing the role that small molecules of diterpene nature play in the stimulation of endogenous neurogenesis through a different mechanism of action, which is the release of growth factors that bind to ErbB receptors through PKC activation. Previous reports show that compounds of this kind do not exert a tumorigenic activity [51] and we have shown that they exert an in vivo effect in animal models [16,17]. A strategy such as the compounds reported in here could be used in combination with recently reported strategies that potentiate differentiation of newly generated neurons in vitro [57]. 2.3. Docking analysis of tested compounds with PKC δ-C1B (PDB: 1PTR) Based on the structural matches between the target compounds in this study and other diterpenes that previously demonstrated similar biological activities, such as phorbol derivatives and ingol-type lathyranes [17,28], and considering that all of them exert their effect through interaction with PKCs, we have considered that it is worthwhile to evaluate ligand/PKC interactions for some selected target compounds. Phorbol esters form the PKC-phorbol complex through hydrogen bonding interactions, primarily with three amino acid residues: Gly-253, Thr-242, and Leu-251. These residues target phorbol esters at specific atoms, resulting in five hydrogen bonds interactions: a) three hydrogen bond donors (HBD) interactions between the C 4 -OH and the main-chain carbonyl of Gly-253, and between the C 20 -OH and the main-chain carbonyls of Thr-242 and Leu-251 and b) two hydrogen bond acceptors (HBA) interactions between C 3 -O and the main-chain amide of Gly253, and between C 20 -OH and the main-chain amide of Thr-242 [58]. Previous results have shown that the capacity of ingol-type lathyranes to promote the differentiation of neuroblast to neurons is related to their ability to activate novel PKCs [16]. Therefore, to explain the differences in the activities observed in the tested compounds, we opted to investigate the potential interaction of selected compounds with the C1B subdomain of novel PKCs. Docking were conducted to reveal the potential binding mode of epoxyboetirane A (5), (8R)-8-hydrox- yeuphoboetirane A (6), and (8R)-8-hydroxyepoxyboetirane A (12) with the PKC δ-C1B subdomain. Molecular modeling studies were based on the X-ray structure of the PKC δ-C1B complexed with phorbol 13-acetate (PDB ID: 1PTR) [58]. Both epoxyboetirane A (5) and (8R)-8-hydrox- yeuphoboetirane A (6) exhibit similar interactions with the C1B subdomain, forming two HBA interactions involving Gln-257 and Thr-242 (Fig. 6A-B and S90A-B). However, 6 differs with an additional HBA interaction, involving Leu-251 and C 8 -O. Their orientation in the enzyme pocket varies slightly, establishing interactions with oxygen atoms at different positions. Specifically, Gln-257 contributes a HB to the C 3 -O in 5 or C 5 -O in 6, while Thr242 forms a HB with the epoxide oxygen situated between C-6 and C-17 in 5 or with the C 8 -OH in 6 (Fig. 6A-B). On the other hand, molecular docking analyses reveal that compound 12, which differentiated NCS into neurons, and phorbol esters bond to similar regions of the PKC δ-C1B domain. Compound 12 engages with the same amino acids and demonstrates three HBD interactions. These interactions occur between the free hydroxyl group at C-8 (C 8 -OH) and the main-chain carbonyls of Gly-253, Leu-251, and Thr242. Additionally, there are two HBA interactions between C 8 -OH and the amine group of Thr242, and between the oxygen of the epoxy function and the hydroxyl group of Thr242 (Fig. 6C and S90C). These results agree with those of biological activity showing that two oxygen atoms located at a distance of 3 Å approximately are necessary to activate the enzyme. 2.4. Molecular dynamics simulation of tested compounds with PKC δ-C1B (PDB: 1PTR) and PKC α -C1B (PDB: 2ELI) To assess the stability of the complexes, molecular dynamics (MD) simulations were conducted for the complexes of compounds 5, 6, and 12 with PKC δ-C1B. The stability of each protein complex during the 100 ns simulation was monitored by plotting the root mean square deviation (RMSD) of the complexes relative to their initial structures, as shown in Fig. 7 and Fig. S91. RMSD measures the deviation of atomic positions and indicates structural changes throughout the MD simulation. The higher and more unstable RMSD values observed for the PKC δ-C1B complexes with compounds 5 and 6 suggest structural instability upon binding with these ligands, as shown in Fig. S91. Additionally, the interaction between (8R)-8-hydroxyepoxyboetir- ane A (12) and PKC α -C1B and PKC δ-C1B was evaluated (Fig. S92). Plots of the potential energy over time are shown in Fig. S93, indicating that all systems in the study were well equilibrated and remained stable throughout the simulation. The stability of each protein complex ((8R)- 8-hydroxyepoxyboetirane A (12) with PKC α -C1B and PKC δ-C1B) during the simulation was also monitored by plotting the RMSD of the complexes relative to their initial structures, as illustrated in Fig 7. The F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 8 Fig. 4. Effect of lathyrane-type diterpenes on the PKC-dependent release of NRG1. A. Schematic representation of mCherry-NRG1-eGFP construct. B. Schematic representation of the mechanisms involved in NRG1-bound fluorescence release. C. Quantitative analysis of the ratiometric microscopic images obtained from the time-lapse assays of HEK293T cells expressing mCherry-NRG1-eGFP and stimulated with 1 µM concentrations of 12, 15, and 16. mCherry/eGFP ratios were normalized to the average mCherry/eGFP ratio measured before stimulation. The mean normalized mCherry/eGFP ratios and SEM are shown, n =40. D-G. mCherry/ eGFP ratio images at the indicated time points are shown in the intensity-modulated display mode. The color range goes from red to blue to represent mCherry/eGFP ratio. The upper and lower limits of the ratio range are shown on the bottom. Scale bar represents 25 μ m. F. Escobar-Monta˜ no et al. Bioorganic Chemistry 153 (2024) 107769 9 and NPT ensemble simulations for 125 ps at 300.15 K, using positional restraints of 400 kJ mol −1 nm −2 on the backbone and 40 kJ mol −1 nm −2 on the side chains. Finally, the complex underwent a production simulation run for 100 ns in the NPT ensemble at 300.15 K and 1 bar. CRediT authorship contribution statement Felipe Escobar-Monta˜ no: Resources, Investigation. Ricardo G´ omez-Oliva: Investigation. Abdellah Ezzanad: Software, Formal analysis, Data curation. Sonia V´ azquez de G´ orgolas: Investigation. David Zorrilla: Software, Formal analysis, Data curation. Antonio J. Macías-S´ anchez: Writing – review & editing, Project administration, Funding acquisition. Jos´ e M. Botubol-Ares: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Conceptualization. Pedro Nunez-Abades: Project administration, Funding acquisition. Carmen Castro: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Funding acquisition, Conceptualization. Rosa Dur´ an-Patr´ on: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Rosario Hern´ andez-Gal´ an: Writing – review & editing, Writing – original draft, Project administration, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This research was co-financed by grants PID2022-142418OB-C22 and PID2022-142418OB-C21, funded by MCIN/AEI/ 10.13039/ 501100011033 and ERDF Operational Program, and by grant P18-RT- 2655, funded by the Department of Economic Transformation, Industry, Knowledge, and Universities of the Regional Government of Andalusia. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioorg.2024.107769. References [1] M.J. Stone, D.H. Williams, On the evolution of functional secondary metabolites (natural products), Mol. 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