*Citation: Ramsay EE and Dilda PJ (2014) Glutathione S-conjugates as prodrugs to target drug-resistant tumors. Front. Pharmacol. 5:181. doi: 10.3389/fphar.2014.00181 This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Ramsay and Dilda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Glutathione levels modulation as a strategy in host-parasite interactions—insights for biology of cancer

#### *Francesco Pennacchio1, Antonio Masi 2 and Alfonso Pompella3 \**

*1 "E. Tremblay" Lab of Entomology, Department of Agriculture, "Federico II" University of Naples, Naples, Italy*

*2 Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padua, Padua, Italy*

*3 Redox Pathology Lab, Department of Translational Research NTMC, University of Pisa Medical School, Pisa, Italy*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Chiranjib Chakraborty, Galgotias University, India*

#### *Reviewed by:*

*George Priya Doss C, VIT University, India*

**Keywords: insect parasitism, bacterial infections, gamma-glutamyltransferase, exosomes, cancer progression**

Parasitic organisms establish a symbiotic association with individuals of a different species—the host—to obtain the metabolic resources needed for their survival and reproduction. These antagonistic associations are characterized by a complex "host-parasite molecular cross-talk," shaped by evolutionary processes largely driven by the requirement of making host's tissues more accessible to a successful colonization and exploitation (Hébert and Aubin-Horth, 2014). In a large number of cases, parasitism not only impairs host physiology and reproduction, but may even culminate with its death or complete consumption. This is the rule in insects, which include the largest number of species with parasitic life habits, characterized by subtle virulence strategies, often shared among organisms belonging to distant phylogenetic groups (Pennacchio and Strand, 2006). The astonishing diversity and the unexpected similarities shared with unrelated taxonomic entities offer the opportunity of insightful comparisons among virulence strategies targeting conserved molecular pathways, as a result of convergent evolutionary patterns.

Here we focus on a comparative analysis of a peculiar strategy of host physiological regulation, targeting the redox homeostasis, which appears to modulate parasitic interactions both at the organism and cellular level.

Insect studies, dealing with parasitism of aphids, have shown that the disruption of host GSH pool and metabolisms significantly contributes to its physiological regulation and castration (Pennacchio and Mancini, 2012). The parasitic wasp *Aphidius ervi* injects at the oviposition into host aphids a venom containing large amounts of a gamma-glutamyltransferase (*Ae-GGT)*, which triggers apoptosis in the upper part of the aphid ovarioles and therefore blocks oogenesis (Falabella et al., 2007). How this selective targeting is achieved and modulated at molecular level is still obscure; however, preliminary data indicate that in parasitized host aphid a depletion of GSH occurs, which primarily involves ovarian tissue (Masi and Pennacchio, unpublished results). It does not require a leap of imagination to speculate that injected *Ae*-GGT can compete with endogenous GGT for substrate GSH, thus interfering with the regular GSH cycle and exposing GSH-depleted cells to an oxidative stress, which ultimately triggers apoptosis. However, it is also reasonable to consider that the alteration of cell survival/apoptosis balance might ensue from the known pro-oxidant effects of GGT activity, due to increased metal-reducing ability of the GSH metabolite cysteinylglycine and extracellular production of reactive oxygen species (Paolicchi et al., 2002). The presence of a GGT in the genome of an entomopoxvirus associated with the parasitic wasp *Diachasmimorpha longicaudata* (Hashimoto and Lawrence, 2005), injected at the oviposition and used as a delivery system of virulence factors, indicates that this enzyme may have additional regulatory roles of host physiology, which remain largely elusive. The possible negative impact on host immunity (Clark et al., 2010) is certainly worth of consideration.

Studies on virulence factors of microrganisms have documented that the invasion strategies of selected pathogenic bacteria also target host GSH metabolism. Indeed, it has been shown that GGT activity of *Helicobacter pylori* and *H. suis,* the agents responsible of peptic ulcer, can exert antiproliferative and pro-apoptotic effects in gastric epithelial cells (Schmees et al., 2007; Gong et al., 2010; Ricci et al., 2014). These effects are triggered by GGT-dependent metabolism of GSH and production of H2O2, which result in the activation of NF-kB, up-regulation of interleukin-8 and increased oxidative DNA damage (Gong et al., 2010; Ricci et al., 2014). Paradoxically enough, supplementation with glutathione of *H. pylori* GGT-treated cells strongly enhanced the harmful effects (Flahou et al., 2011; Zhang et al., 2013). By confocal microscopy, *H. suis* outer membrane vescicles (OMV)—submicroscopic structures 20–50 nm in diamater, budding from the cell surface—were identified as carriers of *H. suis* GGT, capable of delivering the enzyme to the deeper mucosal layers (Zhang et al., 2013). In association with such membranous structures, active GGT from *H. suis* in fact translocates across the epithelial layers and can access lymphocytes residing in the lamina propria of gastric mucosa. The result of this intriguing process appears to be an inhibition of lymphocyte proliferation, i.e., a perturbation of host immunity and a facilitation of bacterial infection (Oertli et al., 2013). Future studies will likely expand the number of examples of GSH-based host-parasite interactions, as possibly in the case of pro-apoptotic activity of GGT released by *Campylobacter jejunii* (Barnes et al., 2007). Cellular GSH appears, thus, to represent a conserved target for parasitic (micro)organisms which aim at altering host redox homeostasis to weaken its immune defenses, using GGT as a key-element of a virulence strategy.

It is possible to further elaborate on this concept by jumping in the field of cancer biology and taking into account the "parasitic" behavior exhibited by malignant cells spreading across tissues and organs of the patient (the "host"). GGT activity is in fact expressed in a number of malignant tumors, and expression levels often increase along with progression of the disease and appearance of more invasive phenotypes (reviewed in Pompella et al., 2006). Importantly, recent studies showed that active GGT can be released from cells, including cancer cells (Franzini et al., 2009), in association with submicroscopic vesicles, 20–40 nm in diameter, resembling exosomes (Fornaciari et al., 2014). The similarity of such structures with GGT-rich OMV particles of *H. pylori* and *H. suis* is indeed obvious. GGT activity of cancer cells can affect intracellular redox equilibria (Pompella et al., 2007), and produces in addition significant extracellular effects, on the S-thiolation status of extracellular proteins (Corti et al., 2005), as well as on the redox status and ligand binding affinity of cell surface receptors related with cell survival/apoptosis balance (Dominici et al., 2004). The question therefore arises, whether GGT-rich exosomes shed by cancer cells can produce in host's surrounding tissues effects comparable to those reported for *Ae*-GGT or *Helicobacter* GGT, possibly resulting in facilitation of malignant cells survival and diffusion.

Collectively, these GSH/GGTdependent processes described in evolutionary distant organisms and different cell populations further corroborate the pivotal importance of redox homeostasis in the modulation of health and disease conditions; shedding light on regulatory elements of these convergent parasitic strategies will likely allow the identification of new potential targets for therapy.

#### **REFERENCES**

Barnes, I. H., Bagnall, M. C., Browning, D. D., Thompson, S. A., Manning, G., and Newell, D. G. (2007). Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni. *Microb. Pathog*. 43, 198–207. doi: 10.1016/j.micpath.200705.007

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 June 2014; accepted: 15 July 2014; published online: 05 August 2014.*

*Citation: Pennacchio F, Masi A and Pompella A (2014) Glutathione levels modulation as a strategy in host-parasite interactions—insights for biology of cancer. Front. Pharmacol. 5:180. doi: 10.3389/fphar. 2014.00180*

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology.*

*Copyright © 2014 Pennacchio, Masi and Pompella. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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**6. Limitations and future directions**

Our research is conceptual in nature. Thus, one of its limitations is that it does not provide an empirical insight into the research model. However, the study presents several opportunities for empirical work. The model should be further explored in future research.

Furthermore, in developing this conceptual model, the researchers did not take into account all factors that may influence green competitive advantage. Instead, they focused on the most important assets.

Another limitation of the study is that this model may be limited to use in companies that have various GHRM practices. In contrast, in Morocco, GHRM practices are not implemented, which hinders the applicability of this model in countries where GHRM is still in its beginning. Thus, these countries may not be able to deploy the proposed framework.

Despite these limitations, the model provides a new starting point for investigating the importance of GHRM in cultivating employees' knowledge, values, and commitment for competitive advantage. We encourage studies to test our proposed model in different countries. Undertaking this research would allow an empirical analysis of whether this conceptual model is applicable in different contexts or not. Finally, future research could investigate this research model in ISO 14001 certified companies or socially responsible companies, as these companies may have already implemented GHRM practices and may recognize its importance.


**5. Discussion**

*Human Blood Group Systems and Haemoglobinopathies*

*4.6.5 Friends and other supporters of Jules*

*4.6.6 The absence of care professionals*

the feeling of abandonment by them.

*4.6.7 The request for recognition of his experience*

outsiders to the family.

he is grateful for this.

beyond his death; unlike the other daughter who died before him. While the dead father remains present (illustrated on the drawing), the sick sister' s absence from the drawing may signify Jules' avoidance of this sister and therefore, avoidance of the threat of death that accompanies her illness. On a psychological level, Jule's drawing may signify that, the living sister is dead, while the dead father remains alive. This complexity of Jules's experiences is illustrated within his drawing.

Jules perceives the children in the neighborhood as helpers and supporters concerning his sister's illness. They are friends who support him by protecting his sister during games and other interactions with her. "*The children do not bother Paulette. They go to school and come back together. They do not cause any problems. Even when we play together, there are no provocations*." Of course, these friends know that Jules is protective of his sick sister. They support him in this way by also protecting her and

Jules' friends are curiously absent from the drawing. He differentiates them from the members of the family. He does not make them part of his family; and hence their absence. In a cultural context where people with whom the subject has special relationships are considered to be members of their family [14], the absence of friends of the drawing is significant. This can tell you that Jules differentiates his friends from family members. Therefore, the support provided by the friends does not compensate for his feeling of abandonment by family members. They are considered outsiders and they may have no obligation over his situation. It could also exhibit his need

Healthcare professionals (hospital and traditional healers) are completely absent

It is also possible that Jules did not draw them because their presence acts as that of the disease and, obviously, the risk of contamination and death. This absence can indicate a psychological effort to avoid anything that reminds him of the disease. In Jules, this absence can refer to the feeling that the traditional therapist does not protect him against the idea of possible contamination by the disease. It is possible that he did not draw the hospital professionals in order to protect himself against

At the end of the drawing, Jules handed his drawing to me while smiling and he asked me to protect it well because it is very fragile. By a projection mechanism, he

At the end of the meeting, with the agreement of Jules and following the requests

of his elder sister, we presented the drawing to her and the mother. It is possible that Jules found by this means a way to make them understand his experience with Paulette's disease in a family context where talking about the disease remains taboo. Particularly moved by finding themselves in the drawing, the elder sister and the mother criticized the "non-human" nature of the characters he drew and the absence of the use of many colors on the drawing. The elder sister put this in relation to the pedagogical approaches used at school, which do not enable children to draw freely.

could be asking me to pay attention to his psychological suffering.

for more support from within as the outside support could be a privilege.

from the drawing. This absence can translate into their investment by Jules as

**50**

With the exception of the sick child who is named by Jules during the interview, parents and siblings are referred to as parents or siblings. The eldest daughter is referred to as "*my big sister*," the brother who is a taxi driver as "*my older brother*," the sick sister as "*the last daughter of mother*," this is very important as she also died of sickle cell anemia. He does not refer to her as 'sister' maybe because he wants to distance himself from the condition which eventually led to her death, the things he fears most, and that have become a part of his family.  Jules refers to her niece as "*the daughter of my older sister*," thereby differentiating his brothers and sisters from his niece. He referred the father and mother as "*father*" and "*mother*."

Four people appear in Jules's drawing. Referred to in the interview as "*my older sister*," the eldest daughter is designated on the drawing as "*the big sister*." The brother who is a taxi driver is, however, named "*big brother Willie*" on the drawing while he is referred to as "*my big brother*" in the interview. The father and the mother are designated in the drawing by "*daddy*" and "*mother*." However, the father is absent from the interview. The absence of the father within Jules' speech is not synonymous with his absence in the psychological universe of the interviewee, as he is present on the drawing. This makes the drawing a mediator to speech and a complementary tool in the interview.

Not all the people mentioned in the interviews appear in the drawing, and those in the interviews are named differently in the interviews. This account for Jules' psychological dynamics concerning his links to family members and the complexity of these links.

The valorization of the traditional treatment (as financially expensive for the families as the medical treatment) contrasts with Jules persistence of the feeling of insecurity regarding wizards. It suggests that the treatment is not necessarily valued for its therapeutic efficacy. In a context where the traditional practitioner is considered as the intermediary between the ancestors and the living [44], it is forbidden to devalue their treatment. To do so would amount to devaluing the ancestors and running the risk of punishment, which could lead to death. The ancestor is incomparable [45] and therefore not criticizable. It can be dangerous to criticize the traditional treatment, as Jules emphasizes that it "alleviates" the fear and concerns the whole family.

Jules did not mention any consultation with a pastor, a priest or an imam. This is a problem in a context where families normally invest religious institutions (churches and mosques) to express their suffering and overwhelming misfortune [46]. It is possible that Jules, whose family is Christian, avoided talking about the care of his sister by the priest or by the pastor to a researcher whose family is of Muslim descent. He would have considered it disrespectful to talk about his religion to a researcher belonging to another religious denomination. It is also possible that, in not telling the researcher about his sister's consultation with priests or pastors, this young boy avoided hitting the researcher's religious sensitivity. He may not have talked to the researcher about this so as not to make him feel uncomfortable during the meeting.

In addition, this study showed that the traditional healer assigns, the uncle rather than the mother in the administration and follow-up of the care of the children and the family. This can be understood by the fact that the woman is often considered by traditional healers, as a being who can reduce the effectiveness of the treatment if she were to touch the patient [47]. Consequently, men are more often empowered to monitor and to enforce the prescriptions of traditional healers. It is also possible that the exclusion of women in this follow-up is motivated by the fact that they are considered to be responsible for the transmission of the disease

to the child [14] and hence as a "stain" in family [4] and a hindrance to traditional treatment. In this case, it would be interesting to understand how mothers and fathers experience the care of their children with sickle cell anemia by hospital and traditional healers.

Jules's family agreed to participate in the research despite the fact that sickle cell anemia remains a taboo subject in African families [2]. It is possible that this family invested the research as a means of understanding the experience of the brothers and sisters of the sick child. The researcher can be considered a mediator who allows the family to talk indirectly about sickle cell anemia with the sick child and his siblings. The parents do not themselves talk with the children about this disease, but they prefer that someone else did it. Although this can be a coping mechanism due to the guilt they experience regarding the situation, they consider themselves as parents who have transgressed a cultural norm whose consequence is the child's illness. This behavior of the parents remains complex. Anyway, the participation of the family in this research shows the interest they had in understanding the experience of all the children in relation to the disease.

This research also allowed Jules to tell the researcher, his family and care professionals his experience with his sister's illness despite it being a taboo subject. It has therefore d upset the norm or violated the family taboo on sickle cell anemia. It would be interesting to understand the impact of Jules and his family's participation in this research on the mother, the sick child and Jules himself.


*4.2. DG*/*DDD*

The ROE-/NOE-interproton distances as well as the RDCs served as input for the distance geometry (DG) and distance bounds driven dynamics (DDD) calculations. The DG pseudo force-field employed for all simulations presented in this study takes the form defined by Equation (1):

$$E\_{\text{total}} = E\_{\text{dist}} + E\_{\text{chir}} + E\_{\text{NOE}} + E\_{\text{RDC}} + E\_{\text{DBL}}.\tag{1}$$

where the dimensionless total pseudo energy *Etotal* is a sum of distance (holonomic bond lengths) errors (*Edist*), chiral volume violations (*Echir*), NOE (*ENOE*), and RDC (*ERDC*) deviations of experimental data from values back-calculated from structures and a special term denoting the deviations of double bonds from planarity (*EDBL*). There are no additional or customary atom-type dependent force-field parameters of physical force-fields used. All pseudo energy terms take the form of sums of squared violations (Δ*X*)2 as defined by Equation (2):

$$E\_X = \frac{1}{2} K\_X \sum \left(\Delta X\right)^2,\tag{2}$$

with Δ*X* = *Xexp* − *Xcalc*, and empirically chosen force-constants *KX* to appropriately account for the size and allowed ranges of each type of parameter violations Δ*X*.

*Edist* and *Echir* (Equation (1)) represent the violations originating from differences in holonomic distances Δ*ri*,*j* (i.e., bond lengths) and Δ*Vi* (chiral volumes). The latter are defined by the scalar triple product *Vchir* = →*a* ·(→*b* × →*c* ) of three vectors spanning planar sp2-type (*Vchir* = 0) or tetrahedral sp3-type atomic centers (i.e., stereogenic centers with *Vchir* - 0), thus encoding for the configuration of the latter through opposite signs ( *V*(*S*) *chir* = *V*(*R*) *chir*), respectively (see Figure 12a). For reference, holonomic distance bounds (for all atom-atom pairs for which upper and lower bounds of inter-atom distances can be established based on the molecular constitution) and chiral volumes are obtained from an initial guess (input) structure of arbitrary configuration and conformation. As these values depend solely on the constitution (which must be known), the DG approach is completely independent from the structure initially assumed [22]. Here, chiral volume restraints were used only on a single stereogenic center simply to avoid enantiomeric structures, as well as on CH3-groups (to keep them tetrahedral) and all sp2-centers (to keep them planar, *Vchir* = 0). Thus, through the deliberate absence of chiral volume restraints, all stereogenic centers (except one), and all CH2-groups with diastereotopic protons were allowed to "*float*" and thus their configurations and/or assignments evolve on the basis of experimental NMR data (NOEs and/or RDCs) only.

In Equation (1), *ENOE* denotes the deviations of back-calculated (and < *r*−6 > averaged where applicable) NOE distances from experimental upper and lower distance bounds with Δ*r* defined as follows:

$$
\Delta r = \begin{cases}
\ r\_{i,j} - r\_{lower} & \text{for} \\
0 & \text{for} \\
\ r\_{i,j} - r\_{upper} & \text{for} \\
\end{cases}
\quad r\_{i,j} \le r\_{i,j} \le r\_{upper}
\quad . \tag{3}
$$

In this study, all NOE bounds *rlower* and *rupper* were derived from the corresponding NMR volume integrals and used as *rmean* ± 10%, with force-constants *KNOE* = 10.0 Å−2 unless stated otherwise.

The mathematics of RDC calculations used here has been taken from Glaser et al. [53], and the formalism on how to include RDC data in 4D and 3D DG simulations (see below) has been described in full detail in Ref. [22]. The harmonic RDC "pseudo energy" *ERDC* is based on violations Δ*D* = *Dexp* − *Dcalc* between experimental and back-calculated values, and RDC data sets derived from multiple alignment media can be used simultaneously as an increasing number of experimental NMR restraints in our *ConArch*+/*DG* approach simply by expanding the corresponding sum in *ERDC*. Empirically, it proved best to scale the force-constant *KRDC* used with the number of RDCs, or equivalently, with the number *M* of alignment media used, and thus we employ *KRDC* = 1.5/*M* Hz−2 in this study.

In addition to the application of chiral volume restraints on sp2-type atomic centers (*Vchir* = 0), the term *EDBL* in Equation (1) is used to reasonably restrain double bonds and aryl rings to planarity (restraining *Vchir* = 0 on neighboring atomic centers alone is not sufficient). Here, Δ*X* (used cf. Equation (2)) is defined as Δ*X* = 1 − cos2 φ, where φ*i*,*j*,*k*.*l* are the corresponding torsion angles *i* − *j* − *k* − *l* with sp2-sp2-type central bonds, and Δ*X* vanishes for φ = 0◦ and φ = 180◦ only (*cis*and *trans*-configurations). The rather high force-constant *KDBL* = 100 used here efficiently removes local energy minima which originate from slight bending of C=C-double bonds and aromatic rings, revealing more distinctive energy steps separating alternate conformational families. In general, the final best-fit energy-minimum structures have very low distortional energy terms of *EDBL* ≤ 5 × 10−3, and the efficiency with which different configurations and conformations are sampled on the basis of experimental NMR data is largely unhampered by these types of restraints.

**Figure 12.** (**a**) Definition of chiral volumes as scalar triple products *Vchir* = → *a* ·( *b* × → *c* ) for sp3- and sp2-type atomic centers. (**b**) In analogy to 2D chiral objects that can be transformed into each other through a rotation in higher dimensions (3D), the configuration of 3D-chiral objects can be inverted by simple rotations in 4D (see text for details). (**c**) Projection mode of higher dimensional objects into lower dimensional space (here visualized as 3D →2D) along the eigenvector associated with the largest eigenvalue λ3 of the inertia tensor (with λ1 < λ2 ... < λ*n* ). Similar projections 4D →3D optimally preserve interatomic distances. (**d**) Temperature dependent scaling factors of projection forces applied to 4D simulated annealing smoothly transforms higher dimensional objects into 3D structures (τ*T* = 150 K, see text for details).

→

The initial input structure is used by DG only for setting up the holonomic bounds and distance matrices (±1% bond lengths), and subsequent configurational and conformational sampling is carried out by our ConArch+/DG approach in an automated sequence of steps. First, molecular structures are generated in four-dimensional (4D) space ("metrization" step, i.e., embedding based on holonomic distance bounds), followed by a 4D "floating-chirality" restrained DG (fc-rDG) and distance bounds driven dynamics (DDD) simulation (simulated annealing). After reduction of dimensionality, the simulated annealing is repeated in 3D space, and each simulation in 4D and 3D is concluded by a gradient-descent type optimization of structures against all restraints, minimizing the total pseudo energy *Etotal*. In all dynamics and optimization calculations, the partial derivatives ∂*Etotal*/∂*r*α of all energy terms with respect 4D and 3D Cartesian atomic coordinates (α ∈ *x*, *y*, *z*(, *w*) for all atoms) are interpreted and used as forces governing the evolution of the system. All derivatives are calculated analytically by ConArch+/DG. During each step of the rDG/DDD runs using RDCs, full updates of the Saupe or alignment tensors are computed based on a singular value decomposition (SVD) algorithm.

Sampling molecular structures first in 4D very e fficiently generates diastereomeric geometries as inversion barriers can be overcome easily [70,71]. Configurational inversion in 3D is reduced to a simple rotation in 4D (see Figure 12b), and consequently during simulated annealing in 4D space with chiral restraints removed on all but selected chiral centers (fc-DDD), the transition barriers between diastereomers are significantly lowered or removed altogether.

For an increased sampling e fficiency, it is crucial to transport as much 4D information as possible into 3D, in order to produce chemically relevant structure models. Projection from higher to lower dimensionality optimally preserves atom-atom distances when carried out along the eigenvector associated with the largest eigenvalue of the inertia tensor *I* defined by Equation (4):

$$I = \sum\_{k} \left( (\overrightarrow{r}\_{k} \cdot \overrightarrow{r}\_{k})E - \overrightarrow{r}\_{k} \bigotimes \overrightarrow{r}\_{k} \right),\tag{4}$$

where the sum runs over all atomic positional vectors ( → *r k* for *k* particles) centered about the origin ( - → *r k* = 0) and weighted with unity mass (see Figure 12c) [72]. During 4D simulated annealing, we apply a temperature dependent scaling factor *f* = exp (−(*T*/τ*T*) 2 ) with an empirical temperature coupling factor τ*T* = 150 K to forces acting along this eigenvector, which gradually restrain the 4D molecular models into a 3D subspace thereof (see Figure 12d). At high temperatures ( *T* > 300 K), all structures evolve freely, but are restrained increasingly and smoothly to 3D sub-space during the cooling phase (*T* < 300 K) of the simulated annealing. Finally, all models are projected into pure 3D space and are subjected to an additional simulated annealing therein.

In this study, for each compound **1**–**5** a total of 1000 structures (configurations and conformations) were generated initially in 4D space. All consecutive simulated annealing simulations in 4D and 3D used 5000 steps of equilibration (*T* = 300 K) and 5000 steps of cooling (*T* → 0 K) each (2 fs time steps). The final structures were collected, sorted by their pseudo energy, and a final selection of the ranked structures of lowest energy were used for the plots presented here. For the global energy minimum best-fit structures, errors in calculated RDCs are estimated from Monte-Carlo bootstrapping analysis including tensor updates [22,23]. Total single processor CPU (Intel(R) Core(TM) i7-4790 CPU @ 3.60GHz) wall time used was about 7–8 min. for each compound **1**–**3**, and up to approx. 50 min for **4** and **5** when three alignment media RDC data sets are used. However, the entire process can be parallelized very efficiently on an arbitrary number of shared memory CPU cores, reducing the total wall time accordingly to a few minutes only.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/6/330/s1, Table S1: NOE data used for Axinellamine A (**1**), Table S2: NOE data used for Tetrabromostyloguanidine (**2**), Table S3: NOE data used for 3,7-*epi*-Massadine chloride (**3**), Table S4: RDC data used for Tubocurarine (**4**), Table S5: NOE data used for **4**, Table S6: RDC data used for Vincristine (**5**), Table S7: NOE data used for **5**, Table S8: Atomic coordinates for rDG structures of compounds **1**-**5**. Figure S1: rDG structure of compound **1**, Figure S2: Extended rDG simulations of **1**, Figure S3: Structures of **1** obtained from extended rDG simulations, Figure S4: rDG structure of compound **2**, Figure S5: Variations of chiral volume restraints used for **2**, Figure S6: Structures of **2** obtained from unrestrained rDG simulations, Figure S7: Structures of **2** obtained from variations of chiral volume restraints, Figure S8: rDG detailed analysis of **2**, Figure S9: Structures of **2** from rDG detailed analysis, Figure S10: rDG structure of compound **3**, Figure S11: rDG detailed analysis of **3**, Figure S12: Structures of **3** from rDG detailed analysis, Figure S13: rDG structure and back-calculated RDCs for **4**, Figure S14: rDG structure of **4**, Figure S15: rDG structure and back-calculated RDCs for **5**, Figure S16: rDG structure of **5**.

**Author Contributions:** Conceptualization, M.K. and S.I.; methodology, M.K. and S.I.; software, S.I.; writing—original draft preparation, M.K. and S.I.; writing—review and editing, M.K., M.R., and S.I.; visualization, M.K. and S.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deutsche Forschungsgemeinschaft (DFG), grant number Re1007/9-1.

**Acknowledgments:** We would like to thank M.R. Scheek (University of Groningen) for providing his modified version of the DG-II program package. We also thank the Center for Scientific Computing (CSC) of the Goethe University, Frankfurt, for granting access to the high-performance computing cluster and providing the CPU time required for the DFT calculations (Figure 11).

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