Design, Synthesis, and Phenotypic Profiling of Pyrano-Furo-Pyridone Pseudo Natural Products

Article abstract of DOI:10.1002/anie.201907853

Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical matter, for instance through biology-oriented synthesis (BIOS). However, BIOS is limited by the partial coverage of NP-like chemical space by the guiding NPs. The design and synthesis of “pseudo NPs” overcomes these limitations by combining NP-inspired strategies with fragment-based compound design through de novo combination of NP-derived fragments to unprecedented compound classes not accessible through biosynthesis. We describe the development and biological evaluation of pyrano-furo-pyridone (PFP) pseudo NPs, which combine pyridone- and dihydropyran NP fragments in three isomeric arrangements. Cheminformatic analysis indicates that the PFPs reside in an area of NP-like chemical space not covered by existing NPs but rather by drugs and related compounds. Phenotypic profiling in a target-agnostic “cell painting” assay revealed that PFPs induce formation of reactive oxygen species and are structurally novel inhibitors of mitochondrial complex I.

Full text of DOI:10.1002/anie.201907853

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Design, Synthesis and Phenotypic Profiling of Pyrano-Furo-
Pyridone Pseudo Natural Products
Authors: Herbert Waldmann, Andreas Christoforow, Julian Wilke, Aylin
Binici, Axel Pahl, Claude Ostermann, and Sonja Sievers
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201907853
Angew. Chem. 10.1002/ange.201907853
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10.1002/anie.201907853
Angewandte Chemie International Edition
Design, Synthesis and Phenotypic Profiling of Pyrano-Furo-Pyridone
Pseudo Natural Products
Andreas Christoforow, Julian Wilke, Aylin Binici, Axel Pahl, Claude Ostermann, Sonja
Sievers and Herbert Waldmann*
[a] M. Sc. A. Chridstoforow, M. Sc. J. Wilke, A. Binici, Prof. Dr. Dr. h.c. H. Waldmann
Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-
Straße 11, 44227 Dortmund, Germany
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
and
Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-
Straße 6, 44227 Dortmund, Germany
[b] Dr. S. Sievers, Dr. C. Ostermann, Dr. A. Pahl,
Compound Management and Screening Center, Dortmund, Otto-Hahn-Str. 11, 44227
Dortmund, Germany.
1
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Abstract
Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical
matter for instance by means of biology-oriented synthesis (BIOS). However, BIOS faces
chemical and biological limitations, due to partial coverage of NP-like chemical and target
space by the guiding NPs. The design and synthesis of “pseudo natural products” (pseudo NPs)
overcomes these limitations by combining NP-inspired strategies with fragment-based
compound design through de novo combination of NP-derived fragments to unprecedented
compound classes not accessible via biosynthesis. We describe the development and biological
evaluation of pyrano-furo-pyridone (PFP) pseudo NPs which combine biosynthetically rarely
related pyridone- and dihydropyran NP fragments in three isomeric arrangements.
Cheminformatic analysis indicates that the PFPs reside in an area of natural product-like
chemical space not covered by existing natural products but rather by drugs and related
compounds. Biological investigation by means of phenotypic profiling in a target agnostic “cell
painting” assay revealed that PFPs induce formation of reactive oxygen species (ROS) and are
structurally novel inhibitors of mitochondrial complex I.
2
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Graphical Abstract.
Phenotypic profiling of pseudo natural products obtained by de novo combination of pyridone-
and dihydropyran fragments reveals a new class of inhibitors of mitochondrial complex I.
3
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Introduction
The discovery of bioactive small molecules with a view to illuminate biology is at the heart of
chemical biology research. For the design of such compound classes, biological relevance is a
key property to consider. One way to guarantee this relevance is to draw inspiration from the
structural properties of the biologically pre-validated molecular repository generated in
evolution.[1] This strategy underlies recently developed approaches like Biology Oriented
Synthesis (BIOS)[2] and the complexity to diversity ring-distortion/modification approach
introduced by Hergenrother et al.[3]
In BIOS, complex natural products (NPs) are simplified to their fundamental core scaffolds for
compound collection design, while characteristic NP properties are retained.[4] However, the
BIOS approach is biased in the exploration of chemical and biological space since it focuses
primarily on the structures of the guiding NPs and their molecular target(s). This focus is
limiting since the currently described NP scaffolds represent only a relatively small fraction of
natural product-like chemical space in a wider sense.[5]
We recently proposed and provided proof for the concept to unite principles of BIOS with the
logic of fragment-based compound discovery[6] to overcome these limitations. In this design
strategy biosynthetically unrelated NP-derived fragments that represent NP structure and
properties[7] are combined de novo to populate NP-like biologically relevant chemical space not
covered by nature. To this end, guiding natural products are deconstructed by cheminformatic
methods[7] to their underlying fragments which then are employed to inspire complexity
generating synthesis routes for their recombination in unprecedented arrangements (Figure 1
illustrates the design approach). The molecular frameworks of such pseudo natural products
(pseudo NPs) are not available from currently utilized biosynthetic machinery and bear the
potential of unexpected and novel bioactivity.[8] In light of this lack of expectable bioactivity,
pseudo NPs will best be evaluated in unbiased target-agnostic cell-based assays monitoring for
instance entire signaling cascades or covering a wide range of biological processes as it is the
case for phenotypic profiling approaches.[9]
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Figure 1: Design of pseudo NPs. NPs are deconstructed to their underlying fragments which
are then recombined in complexity-generating transformations to yield unprecedented
structures not available from current biosynthetic routes.
Here we describe the design and synthesis of a pseudo NP collection obtained by synthetic
recombination of NP-derived pyridone- and dihydropyran fragments to unprecedented pyrano-
furo-pyridone (PFP) pseudo NPs (Figure 2a and b). Analysis of their bioactivity in several cell-
based assays monitoring different biological processes and, in particular, in the multiparametric
“cell painting assay”, which identifies the establishment of complex phenotypes, revealed that
a distinct class of PFP pseudo NPs induce formation of reactive oxygen species (ROS) by
targeting mitochondrial complex I.
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Results and Discussion
For the design of a novel pseudo NP class[6] we intended to combine fragments derived from
biosynthetically unrelated NPs with diverse biological activity to enable reconfiguration of
protein binding patterns. It was planned to combine the fragments in three-dimensional
arrangements including formation of stereogenic centers since stereogenicity correlates
favorably with bioactivity.[6, 10] In addition, it was planned to combine NP fragments with
complementary heteroatoms, in particular nitrogen and oxygen, to create structural difference
from the guiding NPs. With these design criteria in mind we envisioned to combine 2-pyridone-
and dihydropyran fragments which define the structures of numerous NPs with diverse
bioactivities. For instance, the pyridone alkaloid camptothecin (1) is a topoisomerase I targeting
anti-cancer drug[11], and apiosporamide (2) is an antifungal NP[12], whereas for instance the
dihydropyran containing flavonoid pinocembrin (3) and the iridoid catalpol (4) are endowed
with antioxidant[13] and neuritogenic activity[14], respectively (Figure 2a). In addition, an
extensive substructure search in the Dictionary of Natural Products revealed that 2-pyridones
and dihydropyrans are rarely combined in natural products, and if so, only in particular
arrangements (see the Supporting Information, Figure S1).
For the fusion of the pyridone and dihydropyran fragments we chose regioisomerically different
bipodal[6] edge-fused connectivity patterns A-C (Figure 2b) which do not occur in any natural
product. In A-C a linking dihydrofuran fragment with two stereocenters is established which
may be considered a NP fragment on its own[15] (Figure 2b). In addition, the syntheses yielded
compound class D representing a monopodal connection which occurs only in a few NPs (see
the Supporting Information, Figure S1).
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Figure 2: Design and cheminformatic analysis of PFPs. a) Selected NPs incorporating a 2-
pyridone fragment or dihydropyran fragment. b) Design of a pseudo NP collection. The fusion
of functionalized pyridone precursors with functionalized dihydropyran fragments leads to
three PFP pseudo NP classes A-C with bipodal connection between the pyridone- and
dihydropyran fragments, as well as one compound class with mono-podal connection (D). c)
Comparison of NP-likeness scores derived from PFPs (blue curve), DrugBank (orange curve)
and NPs represented in ChEMBL (green curve). d) ALogP vs MW scatter plot of PFPs. 98%
of molecules (158 out of 162 PFPs) fall into Lipinski´s “rule-of-five” space with 61% (99 out
of 162 PFPs) exhibiting lead-like properties. e) PMI plot for PFPs.
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The synthesis of the compound collection commenced with readily available bicyclic 4-
hydroxy-2-pyridones and 4-hydroxy-6-methyl-2-pyridones 5 (Scheme 1), which were obtained
from 4-hydroxy-6-methyl-2-pyrone by treatment with various primary amines[16] (see the
Supporting Information). For the synthesis of functionalized dihydropyrans 7 and 8 (Scheme
1), furyl alcohols 6 were subjected to an Achmatowicz rearrangement[17] to establish the six-
membered 3,6-dihydro-2H-pyran core scaffold. Subsequent protection of anomeric alcohols
with TBSCl, diastereoselective substrate-controlled Luche-reduction of the keto group and
protection of the resulting secondary alcohols as carbonates yielded bis-functionalized
dihydropyran substrates 7 (see the Supporting Information, Scheme S1). Alternatively,
Achmatowicz rearrangement products were only O-acetylated at the acetal oxygen to afford
mono-functionalized dihydropyranones 8 (see the Supporting Information, Scheme S1).
Difunctionalized 3,6-dihydro-2H-pyrans 7 were used as bis-electrophiles in a palladium-
catalyzed allylic alkylation cascade[18] with bis-nucleophilic pyridones 5 to obtain PFPs 9
(general scaffold A, Scheme 1-I). We observed instability for some examples of 9 due to a
Ferrier-like rearrangement[19] of the glycal moiety and decided therefore to perform
heterogenous reduction of the endocyclic double bond to afford isomers 10 (Scheme 1-II) or
exploit the glycal reactivity for further derivatization. For instance, regioselective bromination
of the glycal double-bond[20] and subsequent Suzuki-Miyaura coupling with an electron
withdrawing 4-(trifluoromethyl)phenylboronic acid gave compound 11 (Scheme 1-III).
Furthermore, a direct palladium(II) catalyzed C-glycosidation[21] of 9 with aryl boronic acids
and subsequent isomerization of the pyran double bond into the dihydrofuran ring yielded
compounds 12 (Scheme 1-IV). A Tsuji-Trost alkylation oxa-Michael addition cascade[22]
employing bis-nucleophilic 4-hydroxy-2-pyridones 5 and acetylated dihydropyranone bis-
electrophiles 8 gave PFPs 13 which resemble the general scaffold B (Scheme 1-V). The
corresponding regio-isomers 14 (general scaffold C, Scheme 1-VI) with inverted attachment
points between the pyran fragment and the furopyridone fragment were synthesized by a
quinine-mediated Michael addition-transacetalization cascade reaction[22]. Analogues 15
(Scheme 1-VII) of cyclic acetals 14 in which the methylene-carbonyl substructure is replaced
by a double bond, and their reduced successors 16 (Scheme 1-II), were prepared by an
unprecedented Tsuji-Trost alkylation transacetalization reaction sequence. In addition,
monopodal connection isomers 17 and 18 (Scheme 1-II and -VIII) were synthesized and
employed in the biological analyses. In total, a collection of 162 (+/-)-PFPs were synthesized
in sufficient amounts for biological testing. In general, the compounds were purified
chromatographically (purity >95%) before they were subjected to biological investigations.
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Scheme 1: Synthesis of the PFP pseudo NP library; General reaction conditions: I) R = Me:
Pd(PPh3)4, THF/DMF, rt, overnight, R = H: Pd[P[3,5-(CF3)2C6H3]3]3, THF/DMF, 100-110 °C
MW, 1-2 hours; II) R = H, R = Me: Pd/C, H2, toluene, rt, 1h – 1d; III) 1. NBS, AgNO3,
acetonitrile, 80 °C, 2 hours; 2. Aryl-B(OH)2, NaOtBu, Pd(OAc)2, Xphos, toluene, 130 °C MW,
40 minutes; IV) Aryl-B(OH)2, Pd(OAc)2, DMF, rt, overnight; V) R1 = R2 = H, R1 = R2 = Me, R1
= R2 = N-Boc piperidine, R1 = H R2 = Me: Pd(PPh3)4, NEt3, THF/DMF, rt, overnight; VI) R1 =
R2 = H, R1 = R2 = Me, R1 = R2 = N-Boc piperidine: quinine, DCM, 60 °C, 18 h; VII) R = Me:
Pd[P[3,5-(CF3)2C6H3]3]3, THF/DMF, 100-110 °C MW, 1-2 h; VIII) R1 = R2 = N-Boc piperidine:
HCl in dioxane, 0 °C – rt, 90 min.
To analyze the chemical space occupied by the synthesized 162 PFPs, first the NP-likeness of
the bipodal-fused combinations was evaluated employing the NP-likeness score introduced by
Ertl et al. as a metric tool[23] and compared to NP-scores calculated for molecules listed in
DrugBank[24], and the NP set in the ChEMBL database. The PFP NP scores range in an area of
the graph which is only sparsely covered by NPs (Figure 2c) which reflects that the novel
fragment combinations realized in these bipodal-fused pseudo NPs do not exist in NP scaffolds.
In contrast, the NP-scores calculated for drugs and closely related molecules show substantial
overlap with the majority of the PFP NP-scores, indicating that PFPs may have favorable
physicochemical properties for potential drug discovery programs. Structural relatedness was
further evaluated by mapping estimated hydrophobicity (ALogP) against the respective
molecular weight (MW) using the open-source software LLAMA (Figure 2d).[25] The vast
majority of pseudo NPs (98%, 158 out of 162 PFPs) fall into the Lipinski’s Rule-of-Five
space[26] exhibiting an ALogP < 5 and MW < 500 Da. In addition, 61% of the compounds (99
out of 162 PFPs) fall even into the lead-like space which defines a preferable fraction of
chemical space where ALogP < 3 and MW < 350 Da.[27] Analysis of the three-dimensional
character of the collection and visualization of the shape distribution in a principal moments of
inertia (PMI) plot (Figure 2e)[28] revealed a shift from the linear – disc-like axis towards
spherical shape for the pseudo NPs indicating a three-dimensional character compared to
commercially available compound collections.[25] This observation further shows that the
molecular shape diversity of NPs is conserved in the fragment recombination process.
Collectively, these data suggest that the combination of biosynthetically rarely related
pyridones and dihydropyrans generated novel PFP scaffolds with advantageous
physicochemical properties, increased molecular shape diversity and NP-score distributions not
represented by NPs. This observation mirrors that PFPs are not accessible by biosynthesis such
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that these pseudo NPs define a distinct chemotype which is more than merely the sum of its
fragments eventually representing a distinct area of chemical space. Other NP-fragment
combinations to distinct classes of pseudo NPs were previously reported to follow a similar
trend in the distribution of NP-scores.[6, 29]
Given the unprecedented structure of the pseudo NPs, their bioactivities may be unexpected
and may differ widely from the activities displayed by the guiding NPs. Therefore, similar to
the biological evaluation of newly discovered NPs, they should be investigated in multiple
individual bioassays covering a wide spectrum of biology. Alternatively, pseudo NPs may be
more readily and conclusively characterized by target-agnostic phenotyping approaches based
on high-content technologies,[9] because such phenotypic profiling may efficiently cover larger
areas of biological space in one experimental approach. In agreement with this notion, we
assessed the bioactivity of the PFP pseudo NPs in a „cell painting assay“.[9b, 30] In this multiplex
assay small molecule dyes are employed to selectively stain different cellular compartments in
the absence and presence of compounds. Subsequently, high-content imaging is performed and
automated image analysis extracts and quantifies hundreds of morphological features which are
arrayed in a fingerprint pattern that characterizes the bioactivity of a compound. Comparison
of the obtained morphological fingerprints with the patterns for reference compounds with
annotated bioactivity, may then be employed to generate hypotheses for compound targets or
mode of action and guide subsequent experimental target identification and validation efforts.[9]
Morphological fingerprints for a total of 579 parameters (see the Supporting Information for
the delineation of the parameters) were determined for all PFP pseudo NPs [30d] at 10, 30, and
50 µM concentration and for 3000 reference compounds (mostly at 10µM concentration)
including bioactive small molecules generated in house (see the Supporting Information for
details). For assessment of bioactivity similarity of fingerprint profiles (“biological similarity”,
“BioSim”; see the Supporting Information for determination of similarity) was employed and
in addition an “induction” value (the fraction of parameters (in %) that underwent significant
changes (median absolute deviation (MAD) value upon compound treatment of at least +/-
three-fold of the median determined for the DMSO controls; see the Supporting Information)
was determined as measure for compound bioactivity. This decision was based on the
observation that for the pseudo NP class analyzed here, induction by bioactive members
increased with concentration (see below).
For identification of compounds with probably most interesting bioactivity all PFPs were
considered which at 10 µM showed an induction of at least 10% (bioactivity of compounds
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with lower induction is most likely only low) and not more than 80% (compounds with higher
induction might have multiple targets or pleiotropic activity). This analysis afforded five initial
hits (see the Supporting Information, Table S1). All these hits exhibited a fingerprint profile
similarity of more than 70% to at least one of the reference compound profiles, thus enabling
delineation of biological mode of action or targets.
The obtained hits were clustered according to biological similarity within the hit group itself to
identify compounds with highest potency (i.e. induction) at high biological similarity of at least
80% between two entries (see the Supporting Information for the clustering procedure and
Table S1). The cluster with the pseudo NPs that displayed the highest induction value at 10 µM
contained two members, i.e. Boc protected PFP 14dk (Figure 3a) and the deprotected analogue
14ek (Figure 3a). 14dk and 14ek showed biological similarity of 84% and were structurally
similar (Tanimoto coefficient: 0.72; Table S1). The relatively high biological and chemical
similarity suggest similar modes of action, and these compounds were characterized further.
To prove and justify the choice of induction as a measure for compound bioactivity, the
concentration dependencies of the phenotypes for 14dk and 14ek (Figure 3a) as representative
members of the PFP pseudo NPs were determined by comparing the fingerprints at 10, 30 and
50 µM. For PFP 14dk induction increased from 10 to 30 µM and remained constant at 50 µM,
with biological similarity remaining high (>80%) at all concentrations. For 14ek induction
increased with concentration while biological similarity remained at an average of 66%. Thus,
for 14dk and 14ek induction increased with concentration, while the fingerprint shape remained
comparable with concentration-dependent change of induction, such that induction can be
employed as a measure for bioactivity. The lower profile similarity observed for 14ek at 10 vs.
30 or 50µM probably reflects the lower bioactivity of 14ek as compared to 14dk.
For identification of potential mode of action, a cross correlation analysis for profiles
determined for 14dk and 14ek at 10, 30 and 50 µM, and the set of common found reference
compounds at all measured concentrations was performed to generate a cross-correlation matrix
(see the Supporting Information, Table S3).
For both compounds at 30 and 50 µM, high biosimilarities (> 83%) were found to reference
compounds which inhibit mitochondrial respiration, autophagy, glucose uptake as well as Wnt-
and Hedgehog pathway signaling.[6, 31] Subsequent investigation in cell-based assays excluded
autophagy and Hedgehog signaling as well as glucose uptake inhibition as potential modes of
action. However, we noted that these developmental and metabolism networks are involved in
the regulation of reactive oxygen species (ROS) formation.[32] In addition, the fingerprint
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determined for PFP 14dk at 30 µM showed 88% similarity to the fingerprint determined for
aumitin (19, Figure 3a), a known inhibitor of mitochondrial respiration by targeting
mitochondrial complex I.[31d]
Since these results suggested that pseudo NP 14dk may modulate mitochondrial function as
well, a Mito Stress Test assay was carried out employing the Seahorse XF analyzer as
previously reported.[33] This assay enabled the evaluation of the effect of 14dk on oxygen
consumption rate (OCR) and extracellular acidification rate (ECAR) reflecting the rates of
mitochondrial respiration and glycolysis, respectively (Figure 3b). At 10 and 30 µM, compound
14dk rapidly induced partial inhibition of mitochondrial respiration which the cells
counterbalanced by increasing their glycolysis rate. Given the fact that inhibition of
mitochondrial complexes I and III induces formation of superoxide[34] we assayed formation of
mitochondrial superoxide employing the fluorogenic indicator MitoSOX Red.[31d] Pseudo NPs
14dk and 14ek induced concentration dependent mitochondrial superoxide formation in HeLa
cells after 1 h incubation with EC50 values of 3.7 ± 0.9 µM and 10.7 ± 3.6 µM, respectively (see
the Supporting Information, Table S4 entries 3 and 13).
In the context of formation of reactive oxygen species (ROS) it was reported that ROS is
involved in aging and pathogenesis of several diseases.[35] Furthermore, induction of ROS
formation is considered a potential opportunity for the development of new cancer therapies
through selective enhancement of cancer-cell cytotoxicity.[36]
To further validate the hypothesis of complex I or III inhibition by pseudo NP 14dk, the
previously reported semi-intact assay for mitochondrial respiration was performed.[37] This
assay enables the individual investigation of either complex I-, II-, III-, or IV-mediated
respiratory activity by permeabilizing the cells with a Seahorse XF Plasma Membrane
Permeabilizer (PMP) and adding distinct substrates required for activity of the respective
complexes (Figure 3c).[37] At 30µM compound 14dk partially inhibited complex I as compared
to a full inhibition by the known complex I inhibitor rotenone[38] at 1 µM, while complex II and
IV were not affected by 14dk. For complex III a potentially weak inhibition could not be
excluded as a slight decrease in OCR compared to the DMSO control was observed upon
addition of 14dk. While molecules exhibiting dual inhibitory activity on complex I and III were
already described in the literature[39], the structural elements of 14dk and its fragments do not
resemble any of the three major classes of complex I inhibitors or reported complex III
inhibitors.[40]
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These results demonstrate that target agnostic multiparametric phenotypic profiling, as
implemented in the cell painting assay, may enable determination of mode of action of pseudo
NPs and, by analogy, other bioactive small molecules.[41]
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Figure 3: a) Concentration-dependent fingerprint comparison of 14dk and 14ek (see Table S4,
entries 3 and 13) as well as fingerprint comparison between 14dk and Aumitin (19); The top
line in each bar graph is set as reference fingerprint (100% BioSim) to which subjacent
fingerprints are compared, respectively; blue indicates a decrease of a specific parameter
compared to DMSO control; red indicates an increase of a specific parameter compared to
DMSO control. b) Influence of compound 14dk on the mitochondrial respiration. HeLa cells
were treated with compound and the oxygen consumption rate (OCR) and extracellular
acidification rate (ECAR) were measured with a Seahorse XFp analyzer. Control inhibitors
were added successively to the samples. Data is mean ± SD, n=3. c) Mitochondrial complex
inhibition assay. HeLa cells were permeabilized by Seahorse XF plasma membrane
permeabilizer and treated with 14dk and the respective substrates of the individual complexes.
Control inhibitors were added successively to the samples. Data is mean ± SD, n=3.
Beyond the identification of mode of action, we analyzed the cell painting data to determine
whether this multiparametric phenotypic assay may guide identification of qualitative trends in
structure phenotype relationship (SPR). Subsequently, we assessed if the derived trends in SPR
correlate with trends in structure activity relationship (SAR) determined by means of the
MitoSOX Red assay. These analyses could potentially inform hit expansion through synthesis
of additional compounds. To this end, all pseudo NPs with induction > 10 % and < 80% at all
concentrations (10, 30 and 50 µM) were clustered yielding a 16-membered cluster of
structurally related compounds with biosimilarities > 82% (see the Supporting Information,
Table S2). Since 14dk displayed the highest induction at 10 µM and is a member of this cluster,
its fingerprint profile determined at 10 µM was set as a reference phenotype to which all other
cluster profiles and fingerprints of structurally related compounds were compared. To assure
that the comparison is not negatively influenced by larger differences in fingerprint richness,
comparison was preferably made in an induction range of 20 – 40% (see the Supporting
Information, Table S4). Compounds representing the general trends in SPR were then subjected
to EC50 determination in the MitoSOX Red assay (see the Supporting Information, Table S4).
Comparison of the induction values as measure for bioactivity (see above) revealed that
variation of the pyridone N-substituent at retained bioactivity is possible, but that the substituent
should not be too small (see the Supporting Information, Table S4, compare entries 1-7) or too
polar (Table S4, entries 8-10) to establish higher induction values. Thus, replacement of the
methoxy-indole (Table S4, entry 3) by different substituted phenyl groups or a thiophene led to
high induction at comparable profile similarities (Table S4, compare entry 3 with entries 4-7).
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An isopentyl substituent also resulted in a high induction value, but if a methyl group was
introduced, bioactivity was lost (Table S4, entries 1 and 2). Replacement of the lipophilic
phenyl rings by a basic pyridine (Table S4, entries 8 and 9) abrogated induction, but if a chlorine
was placed next to the basic nitrogen, thereby reducing basicity, bioactivity and biosimilarity
were reestablished (Table S4, entry 10). These observations indicate that for R1 a lipophilic
residue at a distance to the fused ring system is advantageous.
For R2, a bipodal-fused and disubstituted cyclic acetal was beneficial for induction at high
biological similarity to 14dk (Table S4, compare entries 11, 12 and 16 with entries 13 and 14).
In addition, an ,-disubstituted ketone substructure with the quaternary carbon next to the
acetal was required for activity (Table S4, compare entries 13 and 14 with entries 15 and 16).
Fragments of 14dk (Table S5, entries 1-3) were either not active or had low biological similarity
to 14dk. In addition, the central cyclic acetal structure C seemed to be important for activity as
compared to scaffold types A, B and D. This trend was also apparent in a statistical activity
analysis of all substructure classes (see the Supporting Information, Figure S2). However, the
central cyclic acetal scaffold alone did not suffice to establish the observed kind of bioactivity.
Compound 14aa (Table S4, entry 17) representing the core scaffold exhibited almost no
induction even at the highest measured concentration.
The EC50 values determined in the MitoSOX Red assay qualitatively parallel the SPR
conclusions detailed above. Thus, compound 14dk (Table S4, entry 3) was the most potent PFP
pseudo NP with an EC50 value of 3.7 ± 0.9 µM, and most of the compounds with induction
>20% and profile similarity >80% to 14dk induced ROS formation (Table S4, entries 4-7, 10,
13). However, there were some notable exceptions (Table S4 entries 2, 11 and 14), indicating
that, not surprisingly, the SPR-SAR correlation is not fully parallel. While compound 14dc
(Table S4, entry 2) must be regarded as a true outlier exhibiting activity in the cell painting
assay with high bio similarity to 14dk but not in the MitoSOX Red assay, 14cf (Table S4, entry
14) elicited induction but with a profile similarity of 62% to 14dk indicating that the
morphological changes might be induced through a different mode of action. Compound 13ah
(Table S4, entry 11) may be considered a borderline case since induction is 15%, i. e. close to
the 20% cut-off.
Consistent with the SPR, the fragments of 14dk were not active in the MitoSOX Red assay.
Notably, fragment 8d (Table S5, entry 2) deviated from this trend. However, this compound
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induced decreased cell count and cell toxicity at all measured concentrations in the cell painting
assay (see the Supporting Information, Figures S6-8).
Noteworthy, the fingerprints for PFP pseudo NPs could not be simulated by the mathematical
addition of cell painting derived fingerprints of their respective fragments (see the Supporting
Information, Figure S9). This shows that the synthetic combination of NP-fragments generates
pseudo NPs with properties that are more than merely the sum of topologic characteristics of
the individual fragments.
Conclusion
We have designed and developed a synthesis for a new pseudo natural product (pseudo-NP)
class, the pyrano-furo-pyridones (PFPs), that combines two biosynthetically only rarely related
natural product fragments in three different regioisomeric arrangements to arrive at structurally
unprecedented, and therefore biosynthetically not accessed chemical matter. These pseudo NPs
exhibit favorable drug-like features and occupy an area of NP-like chemical space different
from NPs. Unbiased morphological profiling of PFPs in the cell painting assay and subsequent
analysis, based on comparison of phenotypic fingerprints as compared to the fingerprints
determined for annotated reference compounds, guided the discovery that the PFPs define a
structurally novel class of mitochondrial superoxide formation inducers. Analysis of the
morphological profiling data, employing the induction parameter as a qualitative measure for
bioactivity, allowed us to establish general trends in SPR. These could be validated by
determining EC50 values for structurally related compounds in a MitoSOX Red assay. The
observed bioactivity was traced to inhibition of mitochondrial complex I as at least one
responsible molecular target for the activity of the most potent compound.
Our results provide proof-of-principle for the validity of the pseudo NP concept for the de novo
design and synthesis of novel biologically relevant compound classes. Furthermore, they
demonstrate the validity of phenotypic profiling, as exemplified by the cell painting assay, as a
method to determine and characterize potential bioactivity of novel compound classes in a
general sense. Beyond phenotypic profiling, the PFP pseudo-natural product collection was
investigated in individual assays monitoring signaling through the Wnt- and hedgehog
pathways, inhibition of autophagy, indoleamine-2,3-dioxygenase and histone deacetylase
SIRT-1, as well as glucose transport by the GLUT1-4 transporters. However, in none of these
assays was substantial activity recorded at 10 µM concentration which could justify deeper
17
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10.1002/anie.201907853
Angewandte Chemie International Edition
investigation. These findings further highlight the potential value of pseudo NP profiling in
unbiased morphological assays like cell painting, monitoring a wide range of biological
processes simultaneously.
We note that qualitative structure-activity trends, as established here, may not be fully
representative of quantitative SAR analyses. In particular, compounds at an early hit stage as is
certainly the case for the PFPs discussed here, may have multiple targets, and these may differ
among the library members, such that caution is indicated not to over-interpret the data obtained
by the phenotypic analysis. However, if the phenotypic data follow a trend, as is the case for
the PFPs as well, they may guide hit expansion into more potent compounds. They may also
inform structural analysis for instance with respect to identification of a subsite in the
compounds suitable for attachment of reporter groups and linkers which will enable subsequent
true target identification efforts. The different kinds of information that can be obtained from
phenotypic profiling, as shown above and beyond our analysis, will be particularly valuable in
cases where target identification efforts following established methods frequently fail, for
instance if the target protein is expressed only on a very low level, if it is a membrane protein,
or if both conditions coincide.
18
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10.1002/anie.201907853
Angewandte Chemie International Edition
Keywords
Cell-based screening, High-throughput screening, Inhibitors,,Pseudo natural products, cell
painting.
Acknowledgements
Research at the Max Planck Institute of Molecular Physiology was supported by the Max Planck
Society.
Declaration of Interests
The authors declare no competing interests.
19
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10.1002/anie.201907853
Angewandte Chemie International Edition
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