Chemistry & Biology Article · 2016. 12. 1. · Chemistry & Biology Article Engineering the Biosynthesis of the Polyketide-Nonribosomal Peptide Collismycin A for Generation of Analogs

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Chemistry & Biology

Article

Engineering the Biosynthesis of the Polyketide-Nonribosomal Peptide Collismycin A for Generationof Analogs with Neuroprotective ActivityIgnacio Garcia,1 Natalia M. Vior,1 Javier Gonzalez-Sabın,2 Alfredo F. Brana,1 Jurgen Rohr,3 Francisco Moris,2

Carmen Mendez,1 and Jose A. Salas1,*1Departamento de Biologıa Funcional e Instituto Universitario de Oncologıa del Principado de Asturias, Universidad de Oviedo,

33006 Oviedo, Spain2EntreChem S.L, Edificio Cientıfico Tecnologico, Campus El Cristo, 33006 Oviedo, Spain3Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington,

KY 40536-0596, USA

*Correspondence: [email protected]://dx.doi.org/10.1016/j.chembiol.2013.06.014

SUMMARY

Collismycin A is a member of the 2,20-bipyridyl familyof natural products that shows cytotoxic activity.Structurally, it belongs to the hybrid polyketides-nonribosomal peptides. After the isolation andcharacterization of the collismycin A gene cluster,we have used the combination of two different ap-proaches (insertional inactivation and biocatalysis)to increase structural diversity in this natural productclass. Twelve collismycin analogs were generatedwith modifications in the second pyridine ring ofcollismycin A, thus potentially maintaining biologicactivity. None of these analogs showed better cyto-toxic activity than the parental collismycin. However,some analogs showed neuroprotective activity andone of them (collismycin H) showed better valuesfor neuroprotection against oxidative stress in azebrafishmodel than those of collismycin A. Interest-ingly, this analog also showed very poor cytotoxicactivity, a feature very desirable for a neuroprotec-tant compound.

INTRODUCTION

Microorganisms are an important source of bioactive natural

products. About half of them are produced by members of the

actinomycetes family (Berdy, 2005; Butler, 2008; Demain and

Sanchez, 2009; Newman and Cragg, 2007; Olano et al., 2011).

One of the most important groups of bioactive compounds is

hybrid polyketides-nonribosomal peptides. They contain both

polyketide and peptide moieties and therefore multifunctional

enzymes with assembly line-like structures and are responsible

for the biosynthesis of these hybrid compounds (Du and Shen,

2001). In these compounds, usually the polyketide moiety is syn-

thesized by a type I compound (modular polyketide synthase).

Collismycin A (Figure 1A) is a member of the 2,20-bipyridylfamily of natural products that also includes SF2738B-F (Gomi

et al., 1994), pyrisulfoxins (Tsuge et al., 1999), and caerulomycins

1022 Chemistry & Biology 20, 1022–1032, August 22, 2013 ª2013 El

(Funk and Divekar, 1959; McInnes et al., 1977, 1978). Overall,

several biologic activities have been associated with members

of this family of natural products such as antibacterial, antifungal,

and cytotoxic activities (Gomi et al., 1994; Stadler et al., 2001)

and they also have potential as anti-inflammatory agents through

binding to the glucocorticoid receptor (Shindo et al., 1994) or

as neuroprotectants by reducing oxidative stress in neurons

(Martinez et al., 2007). Using labeled precursors, lysine-derived

picolinic acid (PA) has been proposed to be a precursor for the

biosynthesis of these compounds (McInnes et al., 1979; Vining

et al., 1988). Based on their chemical structures, it can be pre-

dicted that they must be synthesized by a hybrid polyketide syn-

thetase-nonribosomal peptide synthetase (PKS-NRPS) system.

This was confirmed through the isolation and characterization

of the collismycin A gene cluster from Streptomyces sp. CS40

(Garcia et al., 2012) and, more recently, with the caerulomycin

gene cluster from Actinoalloteichus cyanogriseus (Qu et al.,

2012; Zhu et al., 2012). With the availability of the sequence of

the collismycin A gene cluster, we were interested in under-

standing how collismycin A biosynthesis takes place and in

generating collismycin analogs with potentially improved thera-

peutic properties. We report a detailed characterization of late

steps in collismycin A biosynthesis. Gene replacement mutants

were generated in seven genes of the cluster, seven analogs

were isolated, and their structures elucidated. In addition, five

other analogs were also produced by biocatalysis. Overall, a pic-

ture on how collismycin biosynthesis takes place has emerged.

We tested the biologic activity of the 12 analogs as cytotoxic

agents, but none of them improved the activity of the parental

compound. However, several analogs showed neuroprotective

activity against oxidative stress when tested in a zebrafish

model, and one of them improved the level of neuroprotection

above that of collismycin A.

RESULTS

Insertional Inactivation of Genes Involved in LateBiosynthesis Steps: Generation of Analogs withModifications in the Substituted Pyridine RingThree phases have been proposed in the biosynthesis of collis-

mycin A (Garcia et al., 2012): (1) biosynthesis and activation of

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Figure 1. Collismycin A

(A) Structure of collismycin A.

(B) Genetic organization of the collismycin A gene

cluster. Gene replacement mutants were gener-

ated in the genes indicated in gray.

Chemistry & Biology

Generation of Collismycin Analogs

PA from L-lysine; (2) formation of the 2,20-bypiridyl, and

(3) tailoring modifications of the 2,20-bipyridyl structure. The

deduced products of several genes of the cluster could be

involved in the third phase of the biosynthesis: clmG1, clmG2,

clmAT, clmD1, clmD2, clmM, and clmM2 (Figure 1B) and would

catalyze enzymatic reactions taking place once the 2,20-bypiridylstructure has been formed.

The pair of genes, clmG1 and clmG2, shows high similarity

with griC and griD from Streptomyces griseus, genes known to

catalyze a carboxylic acid reduction in grixazone biosynthesis

(Suzuki et al., 2007). During the third phase of collismycin A

biosynthesis, the formation of an intermediate carrying a carbox-

ylic group has been proposed (Garcia et al., 2012) and this inter-

mediate could be a good substrate for ClmG1/ClmG2, reducing

the carboxylic group to an aldehyde. Because clmG1 and clmG2

are supposed to act together in collismycin biosynthesis, we

generated a mutant lacking both genes by gene replacement.

Mutant CLM-G1G2 did not produce collismycin A but instead

accumulated a compound (Figure 2) with a mass of 263 m/z

[M+H]. Structural elucidation of the compound revealed a

2,20-bipyridyl family member differing from collismycin A in

possessing a carboxyl group in position 6 and a hydroxyl group

in position 4. We designated this compound collismycin C

(Figure 3). To verify if collismycin C was a real intermediate or a

shunt product, the nonproducing mutant CLM-N1, blocked in

the NRPS gene clmN1 (Garcia et al., 2012), was fed with collis-

mycin C. The mutant was able to convert collismycin C into

collismycin A, suggesting that collismycin C is a real intermediate

(Figure 4A).

The products of genes clmM1 and clmAH were postulated to

act first after the 2,20-bipyridyl ring formation (Garcia et al., 2012).

To confirm this, the previously isolated collismycin SC, a com-

pound accumulated by the CLM-AH mutant (Garcia et al.,

2012), was fed to the CLM-G1G2 mutant. It was found that the

latter mutant was unable to generate collismycin A from collis-

mycin SC. However, almost all collismycin SC was converted

into collismycin C (Figure 4B). These results support a role for

ClmAH as an amidohydrolase, as suggested previously (Garcia

et al., 2012), with collismycin C as the product of the reaction.

The gene clmAT appears to encode a putative aminotrans-

ferase showing high identity with diaminobutyrate-pyruvate

aminotransferases (above 50% identity and above 70% similar-

ity) and with BatP (28% identity and 47% similarity), an amino-

transferase involved in kalimantacin/batumin-related polyketide

biosynthesis in Pseudomonas fluorescens (Mattheus et al.,

Chemistry & Biology 20, 1022–1032, August 22, 2013

2010). These enzymes catalyze the trans-

amination reaction from a donor, usually

an amino acid, to an aldehyde group pre-

sent in the acceptor molecule. ClmAT is

supposed to introduce the nonpyridylic

nitrogen of collismycin A via transamina-

tion. Gene clmAT was inactivated by gene replacement and

the resulting mutant CLM-AT was found not to produce any of

the previously known collismycins, but instead accumulates

two compounds (Figure 2) with masses of 249 and 263 m/z

[M+H]+, respectively. Both compoundswere purified, their struc-

tures elucidated with nuclear magnetic resonance (NMR), and

found to contain a hydroxymethyl group in position 6 but differ

in that the heavier compound contains a methoxy group in posi-

tion 4, while the lighter compound contains a hydroxyl group in

that position. These compounds were designated as collismycin

H and collismycin DH, respectively (Figure 3). Both compounds

were also independently fed to cultures of mutant CLM-N1 to

determine if they were real intermediates (Figures 4C and 4D)

or shunt products. The mutant converted collismycin DH into

collismycins A and H; however, when fed with collismycin H,

no reaction was observed. The presence of a hydroxymethyl

group instead of the expected aldehyde group in position 6 of

collismycins H and DH can be explained by the reactivity of

the aldehyde group, which in the hypoxic cytosolic environment

might be reduced spontaneously to a hydroxymethyl group.

Genes clmD1 and clmD2 of the cluster encode putative dehy-

drogenases that could be involved in oxidative processes during

late steps of collismycin A biosynthesis. The predicted protein

sequence of ClmD1 shows high homology with acyl-CoA dehy-

drogenases from various organisms. The predicted sequence

contains an acyl-CoA dehydrogenase domain typically found in

FAD-dependent oxidases that reduce CoA-bound substrates

(Kim and Battaile, 2002). Inactivation of clmD1 by gene replace-

ment rendered a mutant (CLM-D1 mutant) unable to produce

collismycin A or any related compound (Figure 2). This mutant

can convert collismycin C or collismycin DH into collismycin A

and also produces minor amounts of collismycin H in both cases

(as well as collismycin DH when fed with collismycin C; Figures

4F and 4G). In addition, this mutant can convert collismycin SN

(a compound accumulated by a methyltransferase-minus

mutant, CLM-M1; Garcia et al., 2012). These results suggest

that the reaction catalyzed by ClmD1 occurs early in the biosyn-

thetic pathway, before collismycin SN is formed.

Gene clmD2 encodes a putative FAD-dependent dehydroge-

nase, and the deduced ClmD2 protein shows high similarity with

TamL, a protein involved in oxidative tailoring modifications dur-

ing tirandamycin biosynthesis (Carlson et al., 2010). Although we

have no specific role assigned to ClmD2, the enzyme likely par-

takes in the oxime formation, i.e., late in collismycin biosynthesis.

Inactivation of clmD2 by gene replacement generated mutant

ª2013 Elsevier Ltd All rights reserved 1023

Figure 2. UPLC Analyses of Cultures of Mutants in Different Genes of the Collismycin A Gene Cluster

Mutants were grown for 7 days at 30�C in R5A liquid medium. Cultures were then extracted with either ethyl acetate (wild-type and CLM-AT, CLM-D2-, and CLM-

M2 mutants) or ethyl acetate containing 10% formic acid (CLM-M and CLM-G1G2 mutants). After solvent evaporation, the extracts were analyzed by UPLC. All

chromatograms were extracted at 330 nm with the exception of that of CLM-G1G2 mutant, which was extracted at 280 nm.

Chemistry & Biology

Generation of Collismycin Analogs

CLM-D2, which still produces collismycin A but also accumu-

lates collismycins H and DH (Figure 2). Production of collismycin

A by the CLM-D2mutant and the fact that collismycin DH can be

converted into collismycin A by mutant CLM-N1 (Figure 4C) sug-

gests that ClmD2 might be responsible for oxidizing the hydrox-

ymethyl group in position 5 of collismycin DH to an aldehyde

group, which is the most likely substrate for the aminotrans-

ferase reaction catalyzed by ClmAT. Such an oxidative reaction

would confer ClmD2 the function of recovering the spontane-

ously generated shunt product collismycin DH for the main

pathway. However, we cannot exclude that another dehydroge-

nase present in the cell—although less efficiently—may be able

to catalyze this recovery-reaction.

The clmM gene product shows high similarity with putative

FAD-dependent oxidoreductases and monooxygenases (Garcia

et al., 2012). Because no monooxygenase has been found

to participate in the 2,20-bipirydine formation (Qu et al., 2012),

ClmM could be necessary for late steps of the collismycin forma-

tion, possibly catalyzing the oxime group formation. Upon inac-

tivation of the clmM gene, the resulting mutant CLM-M did not

produce collismycin A, but accumulated a compound (Figure 2)

with a mass of m/z 290 [M+H]+. Structural elucidation of this

compound revealed that it contains a hydroxyl group in position

4 and an N-methylacetamide group in position 6, and was desig-

1024 Chemistry & Biology 20, 1022–1032, August 22, 2013 ª2013 El

nated as collismycin DA (Figure 3). Feeding experiments using

mutant CLM-N1 showed that collismycin DA was not further

modified (Figure 4H), suggesting collismycin DA to be a shunt

product of the pathway, probably generated by acetylation of a

real intermediate by an acetyltransferase present in the microor-

ganism, a common enzyme to detoxify exposed amino groups.

Finally, the gene product of clmM2 is very similar to AzicL, an

O-methyltransferase involved in azicemicin biosynthesis (Oga-

sawara and Liu, 2009). ClmM2 has been proposed to generate

the methoxy group of collismycin A (Garcia et al., 2012). Upon

clmM2 inactivation through gene replacement, the resulting

mutant CLM-M2 did not produce collismycin A, but did accumu-

late three analogs (Figure 2) with masses of m/z 278, 262, and

244 [M+H]+, which were designated as collismycins DS, D, and

DN, respectively. Structure elucidation of these compounds

resulted in the identification of three 2,20-bipyridyl ring-contain-ing molecules with a hydroxyl group in position 4 (Figure 3). Col-

lismycin D and collismycin DN (and also collismycin A) contain a

methylthio substituent in position 5, while collismycin DS con-

tains a methylsulfinyl substituent in that position. However, the

main difference between themwas at position 6, where collismy-

cin D (as well as collismycin A) contains an aldoxime group, while

collismycin DN contains a cyano group in that position, probably

resulting from dehydration of the aldoxime. Nitrile groups have

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Figure 3. Structures of Collismycin Analogs Generated in This Work

See also Figures S1–S26.

Chemistry & Biology

Generation of Collismycin Analogs

been reported forming part of the structure of natural products

(Fleming, 1999; Bezerra Gomes et al., 2010) and a route for the

formation of this nitrile group has been proposed (Olano et al.,

2004). Collismycin DS contains an amido group in position 6,

which could have resulted from a (spontaneous?) hydration of

the cyano group. This type of substituent has also been reported

for other natural products (Bezerra Gomes et al., 2010). Feeding

experiments were carried out with these compounds, again

using the early block mutant CLM-N1, to determine if any of

these were biosynthetic intermediates. (1) Collismycin D was

converted into collismycin A by the mutant (Figure 4I), and a

significant amount of collismycin H was also detected in this

Chemistry & Biology 20, 1022–

bioconversion experiment. (2) The CLM-N1 mutant was also

able to modify collismycin DS, to produce small amounts of an

undetermined compound (indicated by an asterisk in Figure 4J),

which was not purified. However, high-performance liquid chro-

matography/mass spectroscopy (HPLC/MS) findings suggest

the presence of a methoxy group at position 4. (3) The CLM-

N1 mutant was unable to modify collismycin DN (Figure 4K).

Generation of Acylated Analogs of Collismycin H byBiocatalysisCollismycin H (Figure 3) was the major compound accumu-

lated by CLM-AT mutant. The primary hydroxyl group of this

1032, August 22, 2013 ª2013 Elsevier Ltd All rights reserved 1025

(legend on next page)

Chemistry & Biology

Generation of Collismycin Analogs

1026 Chemistry & Biology 20, 1022–1032, August 22, 2013 ª2013 Elsevier Ltd All rights reserved

Table 1. Antiproliferative Assay of the Different Collismycin A

Analogs against a Panel of Tumor Cell Lines

Compound

Cell Line

NIH373

Fibroblast

A549

Lung

MDA-MD-231

Breast

HCT-116

Colon

Collismycin A 56.6 0.3 >100 0.6

Collismycin D 14 4.3 >100 4.8

Collismycin DN >100 >100 >100 >100

Collismycin DS >100 1.9 >100 1.7

Collismycin H >100 >100 >100 >100

Collismycin DH >100 90 100 >100

Collismycin DA >100 41.3 >100 6.6

Collismycin DC >100 >100 >100 >100

Collismycin H1 >100 63.5 >100 53

Collismycin H2 >100 >100 >100 >100

Collismycin H3 >100 84.7 >100 79.4

Collismycin H4 >100 >100 >100 >100

Collismycin H5 >100 >100 >100 >100

A fibroblast cell line was used as toxicity control; values represent IC50 in

micromoles.

Chemistry & Biology

Generation of Collismycin Analogs

compound was considered a good target to generate analogs

by enzymatic acylation. There are reports in the literature

describing acyltransferases with broad substrate specificity

(Xie et al., 2006). With this aim in mind, we chose a biocatalytic

acylation reaction, a greener alternative to conventional chem-

ical acylating reagents. We used lipase PS from Burkholderia

cepacia as biocatalyst and various esters (acetate, chloroace-

tate, butyrate, benzoate, and crotonate) as acyl donors. The

biotransformation with these acyl donors efficiently rendered

collismycin H analogs, carrying the corresponding acyl resi-

dues (Figure 3), namely acetyl (collismycin H1), chloroacetyl

(collismycin H2), butyryl (collismycin H3), crotonyl (collismycin

H4), and benzoyl (collismycin H5). The incorporations of the

corresponding acyl groups were verified with NMR and MS

of the product compounds (see Supplemental Experimental

Procedures).

Antiproliferative Activity of the Collismycin AnalogsCollismycin A and the 12 analogs were tested for in vitro inhibi-

tion of growth of three different tumor cell lines: A549 (lung),

HCT116 (colon), and MDA-MB-231 (breast) using as a control

for toxicity the NIH373 fibroblast cell line (Table 1). Collismycin

A showed half-maximal inhibitory concentration (IC50) values of

0.3, 0.6, and > 100 mM against the three cell lines, respectively.

Both collismycin A and the 12 analogs were poorly active against

the fibroblast cell line. Some of the compounds showed

IC50 values higher than or closer to 100 mM (collismycins H,

DH, DC, DN, H2, H3, H4, and H5), and collismycin H1 showed

IC50 values ten times higher than those of collismycin A. Three

analogs, collismycins D, DA, and DS, showed IC50 values close

to those of collismycin A.

Figure 4. UPLC Analyses of Bioconversion Experiments

Cultures of CLM-N1 mutant (A, C, D, H, and I–K), CLM-G1G2 mutant (B), and CL

indicated in each panel. After 6 days of incubation at 30�C, cultures were extracte

were extracted at 330 nm.

Chemistry & Biology 20, 1022–

Neuroprotective Activity of the Collismycin AnalogsThe neuroprotective action of collismycin A had been previously

reported (Martinez et al., 2007). We tested the collismycin ana-

logs as potential neuroprotectants using an in vivo zebrafish

model by determining induction of apoptosis in neural cells

and potential protection with these analogs. Three days postfer-

tilization (dpf), zebrafish larvae were incubated with all-trans ret-

inoic acid (RA) as a stress inducer in the presence or absence of

collismycin A and analogs. Then, apoptotic cells were observed

with fluorescence microscopy (Parng, 2005; Parng et al., 2007;

Selderslaghs et al., 2009). Control zebrafish larvae, with no RA

treatment, exhibited few or no apoptotic cells, while RA-treated

zebrafish exhibited a significant increase in apoptosis in the brain

region (Figure 5A). As a positive neuroprotectant control, lipoic

acid (LA) was used. Pretreatment of the larvae with LA reduced

apoptosis in approximately 61% at 1 mM. Zebrafish larvae

treated with 1 mM collismycin A were reduced the appearance

of apoptotic cells approximately 44% (Figure 5B). One of the

12 analogs (collismycin H) showed significantly improved neu-

roprotective activity compared to collismycin A, with a 60%

decrease in apoptosis induction (Figure 5B). Another four com-

pounds, collismycins D, DN, DS, and H1, also showed neuropro-

tective activity but to a similar or lower extent than collismycin A,

with 32%, 39%, 36%, and 44% apoptosis reductions, respec-

tively (Figure 5). The remaining six compounds did not show

any significant neuroprotective effect (see Supplemental Exper-

imental Procedures).

DISCUSSION

Collismycin A contains a two-pyridine ring system that is

biosynthesized from lysine involving NRPS and hybrid PKS-

NRPS multi-enzyme machineries. Two biosynthetic intermedi-

ates, collismycin SN and collismycin SC, were isolated after

insertional inactivation of two genes of the cluster (Garcia

et al., 2012). Further gene inactivation experiments, followed

by the isolation of the resulting mutant products, yielded seven

different collismycin analogs with minor chemical modifications

at C4, C5, or C6 positions of the substituted pyridine ring

(described in this manuscript). These results along with feeding

experiments of the mutant products to an early block mutant

allowed us to propose a pathway for the late steps of collismy-

cin A biosynthesis (Figure 6). In this pathway, some of the ana-

logs are proposed to be biosynthetic intermediates while others

are suggested to be ‘‘shunt products’’ generated by some

enzymes of the cluster but not acting in the proper order of

action. The action of amidohydrolase ClmAH would remove

the extra leucine residue added during the biosynthesis, gener-

ating collismycin C (isolated in this work), which contains a

carboxyl group at position 6. The enzyme pair ClmG1-ClmG2

constitutes of a carboxylic acid reductase, which would reduce

the carboxyl group to an aldehyde group. However, we have

been unable to isolate the aldehyde intermediate. Inactivation

of aminotransferase ClmAT, supposedly responsible for the

M-D1 mutant (E–G) were fed with 50 mM of different collismycin A analogs as

d with ethyl acetate/formic acid 1% and analyzed with UPLC. Chromatograms

1032, August 22, 2013 ª2013 Elsevier Ltd All rights reserved 1027

Figure 5. Neuroprotective Action of the Collismycin Analogs

(A) Induction of apoptosis in a zebrafish model as visualized by fluorescence microscopy. The zebrafish larvae were treated with 10 mM RA alone as a stress

inducer (RA control) or in combination with 1 mM of the different collismycin analogs. As a positive neuroprotectant control, zebrafish were treated with

10 mM RA and 1 mM LA. See also Table S2.

(B) Effect of the different collismycin analogs on RA-induced apoptosis in zebrafish larvae. Apoptosis induced by RA in the presence or absence of different

collismycin analogs was determined with fluorescence microscopy and, after quantification, relative apoptosis was standardized using the level of apoptosis

generated by the RA treatment as 100%. One-way ANOVA and Dunnett’s t test were used to determine the significance. A p value less than 0.05 was

considered statistically significant. All analogs exhibited significant protection with p < 0.05. See also Table S1.

See also Figures S27–S29.

Chemistry & Biology

Generation of Collismycin Analogs

transamination of this aldehyde group, accumulated two com-

pounds containing a hydroxymethyl group in position 6 but it

did not accumulate the aldehyde intermediate. The sequential

reduction of oximes to alcohols mediated by alcohol dehydro-

genases has been reported, reactions in which only traces of

aldehydes could be detected (despite being the obvious

precursor; Ferreira-Silva et al., 2010). This suggests that the

reduction of the aldehyde is faster that the aldoxime reduction.

In our case, it is something similar; the reduction of the alde-

hyde is faster than the corresponding carboxylic acid reduction.

The two isolated compounds from CLM-AT mutant, collismycin

H and DH, only differing in the methylation state at position 4,

could be derived from the nonisolable aldehyde intermediate

by spontaneous reduction (and further methylation by ClmM2

in the case of collismycin H). The role of dehydrogenase

ClmD2 in the biosynthesis of collismycin A is therefore not

totally clear. Apparently, its participation is not absolutely

essential for collismycin A biosynthesis, because inactivation

of clmD2 does not prevent formation of collismycin A. However,

this mutant also accumulated collismycin H and collismycin DH,

which prompted us to propose that the action of ClmD2 could

be important for the producer organism, to recycle a spontane-

ously formed shunt product, collismycin DH, into the main

pathway, thereby diminishing the pathway shunt toward collis-

mycins DH and H.

The main pathway continues with aminotransferase ClmAT,

responsible for the transamination of the aldehyde group. So

far, we have not been able to isolate the resulting amine

1028 Chemistry & Biology 20, 1022–1032, August 22, 2013 ª2013 El

analog itself that should be accumulated by the CLM-M

mutant. Instead, this mutant accumulates an acetylated deriv-

ative (collismycin DA) of the expected amine compound,

which was shown to be a shunt product, because it is not con-

verted into collismycin A when fed to early block mutants.

The formation of collismycin DA is probably caused by an

undetermined acetyltransferase of the producer organism,

whose corresponding gene is located outside the clm gene

cluster.

Clearly, methyltransferase ClmM2 is responsible for the

O-methylation of the 4-OH group. Inactivation of clmM2 pro-

duced a mutant that accumulated three compounds, all of

them lacking the 4-methoxy group, thereby pinpointing ClmM20srole for this O-methylation step. Two of the accumulated com-

pounds, collismycin DN and collismycin DS, also possess mod-

ifications at positions 5 and 6. None of them is a biosynthetic

intermediate, because they were not converted into collismycin

A by any of the mutants.

All seven collismycin analogs contain chemical modifications

in the substituted pyridine ring. By using a biocatalytic approach,

we also generated five additional analogs in which, using as

starting material collismycin H, we introduced different acyl moi-

eties (acetyl, chloroacetyl, butyryl, crotonyl, or benzoyl) at the

hydroxyl group of collismycin H.

Overall, 12 collismycin analogs were produced using these

two each other complementing pathway-bioengineering str-

ategies. All were initially tested for their cytotoxic activity

against a panel of three tumor cell lines. None of them showed

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Figure 6. Proposed Biosynthetic Pathway for Collismycin A Biosynthesis

Chemistry & Biology

Generation of Collismycin Analogs

a clear improvement in antiproliferative activity compared to

collismycin A, although three of the analogs (collismycins D,

DA, and DS) showed a cytotoxicity comparable to that of

collismycin A. Some conclusions can be drawn regarding

structure-function relationships. First, the presence of the

oxime moiety at the substituted ring seems to be important

for the cytotoxicity, although it can be replaced by a carbamoyl

moiety (as in collismycin DS), which has similar H-bonding

features. Second, the cytotoxic activity is not affected by the

methylation of the hydroxyl group at C4 of the substituted

ring, because analogs containing a 4-hydroxyl (e.g., collismy-

cins D and DS) or a 4-methoxy group (e.g., collismycin A) are

equally potent.

Zebrafish has been used as a model organism to determine

the toxicity of compounds, also for detecting environmental

hazards and for risk assessment (Chiu et al., 2008; Parng

2005; Tran et al., 2007). In addition, a zebrafish assay for in vivo

neuroprotective action has been developed (Parng et al., 2006).

We have used this model to assay the potential neuroprotective

activity of the collismycin analogs by determining apoptosis

induction caused by RA. One of the compounds tested,

collismycin H, showed enhanced neuroprotective activity when

compared to collismycin A. In addition, collismycin H showed

similar values of neuroprotection as those of the positive control

using lipoic acid and better neuroprotective values than collis-

mycin A. Interestingly, collismycin H showed very poor cytotoxic

activity. These two combined features are important for a com-

pound to be developed for potential clinical use: high efficiency

as neuroprotectant and very low cytotoxicity. Collismycin H

Chemistry & Biology 20, 1022–

lacks the oxime group that seems to be important for the cyto-

toxic activity. On the other hand, the hydroxymethyl group at

C6 of collismycin H can be used as a handle for further minor

modifications (such as acetylation) without losing all neuropro-

tective activity, but it cannot be drastically modified by introduc-

tion of longer acyl groups, because some of the acylated

analogs, such as collismycins H2, H3, H4, and H5, lost the

neuroprotective ability.

Although the mechanism for collismycin cytotoxic and neuro-

protective activities is still unknown, it is possible to speculate

that they could be related to the ability to chelate metals, a

common property to the 2,20-bipyridyl family of compounds.

The 2,20-bipyridyls form complexes with several divalent metal

ions, especially copper and iron (Nakouti et al., 2012; Nocentini

and Barzi, 1996), and it is widely known that, because tumor cells

have higher requirements of iron due to their increased prolifer-

ation rate, treatment with iron chelators is an interesting

approach for cancer treatment (Kovacevic et al., 2011). Chela-

tors not only cause an arrest in DNA synthesis, they can also

affect the expression of key molecules involved in cell cycle

control and angiogenesis or directly generate oxidative stress

through the formation of redox iron complexes (Kovacevic

et al., 2011; Richardson, 2005). The affinity for iron of members

of the 2,20-bipyridyl family seems to be due to the pyridyl nitrogen

atoms and sometimes can be enhanced by the presence of a

third atom, such as the sulfur of the cytotoxic compound 2,20-bipyridyl-6-carbothioamide (Antonini et al., 1981). Collismycin

A could behave as a tridentate ligand, too, with the third atom

being the nitrogen of the oxime. This could explain why, in the

1032, August 22, 2013 ª2013 Elsevier Ltd All rights reserved 1029

Chemistry & Biology

Generation of Collismycin Analogs

collismycin analogs lacking the oxime moiety, the cytotoxic

activity is reduced, but the loss is less severe when a nitrogen

atom remains in that part of the molecule (i.e., collismycins DS

and DA). In the case of collismycin H, the replacement of the

oxime by a hydroxymethyl resulted in a drastic reduction of its

cytotoxic activity, while in contrast its neuroprotective activity

was improved.

Zebrafish neuroprotection assays against oxidative stress-

induced apoptosis have allowed the identification of neuropro-

tective compounds, such as free radical scavengers like

D-methionine, the steroid hormone cortisone, or iron chelators

like desferoxamine (Parng et al., 2006). These compounds, being

structurally quite diverse, probably exert their neuroprotective

role acting on different targets within the cell. There are com-

pounds that directly neutralize the formation of reactive oxygen

species, while others avoid their formation specifically by inacti-

vating enzymes involved in oxidative processes (Youdim et al.,

2004). In the case of collismycin A and its analogs, the fact that

the presence of the oxime seems to be essential for the cytotoxic

activity, whereas it does not affect the neuroprotective potential,

could indicate that these two activities are accomplished

through different mechanisms.

In summary, the combination of two different approaches

(insertional inactivation and biocatalysis) is a tool to increase

structural diversity in the 2,20-bipyridyl family of natural products.

Minor modifications in the collismycin A molecule contributed to

generate analogs possessing very low cytotoxic activity but

acquiring neuroprotective properties against oxidative stress

in zebrafish. This way, genetic engineering of the biosynthetic

pathway of an antitumor compound has rendered potential

analogs with potential as neuroprotectants.

SIGNIFICANCE

Genetic engineering is a powerful tool for the generation

of derivatives of bioactive natural products. A number of

derivatives from different natural products synthesized by

microorganisms have been generated by combinatorial

biosynthesis. The use of this technology and its application

to the biosynthesis of the hybrid polyketide-nonribosomal

peptide collismycin A, a member of the 2,20-bipyridyl family,

together with a biocatalytic strategy allowed the generation

and isolation of 12 collismycin A derivatives. Some of these

compounds were accumulated bymutants in specific genes

of the cluster and the isolation and elucidation of their

chemical structures proposed a biosynthetic route to collis-

mycin A. Some of these derivatives showed potential neuro-

protective activity against oxidative stress in a zebrafish

model while in contrast they lack cytotoxic activity tested

against a panel of tumor cell lines. These two features are

expected for a compound to be potentially developed as

neuroprotectant.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Culture Conditions, and Vectors

Streptomyces sp. CS40, Streptomyces albus J1074, Escherichia coli DH10B

(Invitrogen), and E. coli ET12567 (pUB307; Kieser et al., 2000) were used in

this work. Plasmids pCR-Blunt (Invitrogen), pHZ1358 (Sun et al., 2009),

1030 Chemistry & Biology 20, 1022–1032, August 22, 2013 ª2013 El

pEM4T (Menendez et al., 2006), pUO9090 (Prado et al., 1999), and cosmid

cos1c3 (Garcia et al., 2012) were also used. All Streptomyces strains were

grown in specific media either for growth or production as detailed in the

Supplemental Experimental Procedures.

DNA Manipulation and Mutant Generation

DNA manipulation followed standard procedures (Sambrook et al., 1989).

Plasmid introduction into Streptomyces sp. CS40 or into S. albus was carried

by intergeneric conjugation using E. coli ET12567 (pUB307) as DNA donor

(Kieser et al., 2000). All collismycin nonproducing mutants were generated

by gene replacement. Two approximately 1.5 kb flanking regions for each

gene were obtained with PCR or enzymatic digestion and they were cloned

upstream and downstream from the apramycin resistance gene aac(3)IV

in pUO9090. Then the replacement cassette was subcloned into the

conjugative vector pHZ1358 and the resultant replacement plasmids were

introduced into Streptomyces sp. CS40 and thiostrepton-sensitive, apramy-

cin-resistant clones were selected. The occurrence of the gene replacement

was verified by Southern blot hybridization. Detailed description of the

generation of each mutant is provided in the Supplemental Experimental

Procedures.

Analysis and Purification of Compounds and Structural Elucidation

Cultures of the different strains were analyzed by ultra-performance liquid

chromatography (UPLC) and HPLC-MS for the presence of collismycin or

analogs. Ethyl acetate extracts of whole cultures were analyzed by reversed

phase chromatography, as detailed in the Supplemental Experimental

Procedures. Collismycin analogs were purified from liquid cultures in R5A

medium. Purification was carried out by a combination of solid-phase

extraction and preparative HPLC, as detailed in the Supplemental Experi-

mental Procedures. Structural elucidation of the 12 collismycin analogs

was determined with MS and NMR spectroscopy (see Supplemental Exper-

imental Procedures).

Bioconversion Experiments

Bioconversion assays were carried out in 24-square-deep well plates

(Enzyscreen). Streptomyces sp. mutant strains were grown as seed culture

in 3 ml of Tris-buffered saline. After 24 hr of growth in an orbital shaker

(30�C/250 rpm), 0.1 ml of each culture was used to inoculate 2 ml of R5A sup-

plemented with 50 mMof collismycin analogs; after 6 days (30�C/250 rpm) 2ml

of cultures were extracted with 2ml ethyl acetate/formic acid 1%and analyzed

with UPLC.

Generation of Analogs by Biocatalysis

Ester derivatives of collismycinHwere obtainedbymeans of a lipase-catalyzed

acylation. Lipase PS from Burkholderia cepacia (Amano) was chosen as the

biocatalyst, and vinyl acetate, vinyl chloroacetate, vinyl butyrate, vinyl benzo-

ate, and vinyl crotonate were tested as acyl donors. Reactions were assayed

at 30�C and 250 rpmdissolving 5mg of collismycin H in 1ml of THF and adding

a 3 molar-fold of acylating agent and 10 wt % of enzyme. The lipase efficiently

catalyzed all the processes (conversion degree higher than 95% after 24 hr)

and, after purification by preparative HPLC, rendered collismycin H derivatives

with the corresponding acylated moieties.

Antiproliferative Activity

The cytotoxic activity of compounds was tested against three tumor cell lines:

A549 (lung), MDA-MB-231 (breast), and HCT116 (colon), using a fibroblast cell

line (NIH373) as control. Cells were incubated in 96-well plates (5,000 cells per

well) in 100 ml DMEM medium (Sigma) supplemented with 10% fetal bovine

serum (Biochrom) and 2 mM glutamine. After 24 hr, compounds (dissolved

in Dulbecco’s modified Eagle’s medium [DMEM]) were added at different

concentrations. As cell viability and as cell death controls, DMEM and 0.1%

SDS were added, respectively. After 24 hr of incubation, WST-8 reactive

(Cell Counting Kit-8, Sigma) was added and incubated for 2 hr at 37�C and

then absorbance at 450 nm was determined in an EL3 800 microplate reader

(BioTek). Dose-response curves and IC50 values were obtained using Reader-

fit Online Software (Hitachi).

sevier Ltd All rights reserved

Chemistry & Biology

Generation of Collismycin Analogs

Neuroprotective Activity

The potential of the collismycin analogs as neuroprotectants was tested in an

in vivo zebrafish model through the treatment of 3 dpf zebrafish larvae with

all-trans RA as the stress inducer alone or cotreatedwith the different collismy-

cin analogs. Apoptosis induction was then evaluated by acridinium chloride

staining and observation with fluorescence microscopy (see Supplemental

Experimental Procedures for details).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

29 figures, and two tables and can be found with this article online at http://

dx.doi.org/10.1016/j.chembiol.2013.06.014.

ACKNOWLEDGMENTS

This research was supported by a grant (to J.A.S.) from the Spanish Ministry of

Science and Innovation (BIO2009-07643) and the Plan Regional de Investiga-

cion del Principado de Asturias (PC10-05). N.V. was the recipient of a predoc-

toral fellowship of the Spanish Ministry of Science and Innovation (MICINN,

FPI). We are very grateful to Carlos Olano for helpful discussions. We would

like to thank Marcos Garcia Ocana and ZFBiolabs (Madrid, Spain) for helping

with the cytotoxicity and zebrafish assays, respectively.

Received: April 9, 2013

Revised: June 20, 2013

Accepted: June 25, 2013

Published: August 1, 2013

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