Structural Insights into the Development of Cycloguanil Derivatives as

Subscriber access provided by Stockholm University Library

Article

Structural Insights into the Development of Cycloguanil Derivatives as Trypanosoma brucei Pteridine Reductase 1 Inhibitors Giacomo Landi, Pasquale Linciano, Chiara Borsari, Claudia P. Bertolacini, Carolina Borsoi Moraes, Anabela Cordeiro-da-Silva, Sheraz Gul, Gesa Witt, Maria Kuzikov, Maria Paola Costi, Cecilia Pozzi, and Stefano Mangani ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00358 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

Structural Insights into the Development of Cycloguanil Derivatives as Trypanosoma brucei Pteridine Reductase 1 Inhibitors

Giacomo Landi1, Pasquale Linciano2, Chiara Borsari2†, Claudia P. Bertolacini3, Carolina B. Moraes3, Anabela Cordeiro-da-Silva4, Sheraz Gul5, Gesa Witt5, Maria Kuzikov5, Maria Paola Costi2, Cecilia Pozzi1*, and Stefano Mangani1*

1Department

of Biotechnology, Chemistry and Pharmacy – Department of Excellence 2018-2020,

University of Siena, via Aldo Moro 2, 53100 Siena, Italy 2Department

of Life Science, University of Modena and Reggio Emilia, via Campi 103, 41125

Modena, Italy 3National

Laboratory of Biosciences, National Center for Research in Energy and Materials,

Campinas – SP, 13083-970, Brazil 4Instituto

de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular

and Cell Biology, 4150-180 Porto, Portugal 5Fraunhofer

Institute

for

Molecular

Biology

&

Applied

Ecology

–

ScreeningPort,

Schnackenburgallee 114, D-22525 Hamburg, Germany †Present

address: Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel,

Switzerland *Authors

for

correspondence:

Cecilia

Pozzi,

[email protected]

[email protected]

1

ACS Paragon Plus Environment

and

Stefano

Mangani,

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

Cycloguanil is a known dihydrofolate reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was develop, resulting in 1 and 2a having IC50 of 692 and 186 nM, respectively, towards TbPTR1. Structural analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of hydrophobic interactions, H-bonds and halogen bonds. Moreover, in vitro cell growth inhibition tests indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1 activity in T. brucei is a new promising strategy for the treatment of human African Trypanosomiasis. On this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.

Cycloguanil; triazine derivatives; Trypanosoma brucei; anti-parasite drugs; pteridine reductase; folate pathway.

2


Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Protozoan parasites belonging to the Trypanosoma and Leishmania species are the etiological agents of various neglected tropical diseases, including human African Trypanosomiasis (HAT, also known as sleeping sickness), Chagas disease and different forms of leishmaniasis1. HAT is caused by the bloodstream form of Trypanosoma brucei (T. brucei, or Tb)1. Trypanosoma cruzi (T. cruzi), an obligate intracellular parasite that invades various internal organs, is the etiological agent of Chagas disease. Leishmania spp. infect macrophages and cause a wide spectrum of symptoms ranging from cutaneous lesions to potentially fatal visceral infections2. Around 10 million people worldwide are infected by these parasites. Since effective vaccines against these diseases have not been developed, their treatment relies on chemotherapy and vector control. A very limited number of drugs is in use and most of them are toxic, poorly effective and their clinical efficacy is compromised by the evolution of drug resistant parasites, therefore new therapies are required3,4. In the field of HAT, the introduction of the nifurtimox-eflornithine combination therapy5 allowed a step forward towards the eradication of the infection. It is hope that the introduction of fexinidazole, the first oral drug for the treatment of this disease, will contribute further to the aim6,7. However, drug resistance problems may always occur and the existence of animals that bear the infection8 justifies further the research in the field. The target-based drug-discovery approach represents a promising strategy and it has been successfully applied to develop eflornithine, an ornithine decarboxylase inhibitor, which is currently in use as a therapeutic for the late-stage HAT9,10. Although other targets such as cathepsin L11, trypanothione reductase12, N-myristoyl transferase13, and farnesyl transferase14 have been validated for T. brucei, no drugs acting on them have been approved for the treatment of HAT. Enzymes of the folate pathway are established targets for bacterial infections15 and malaria16 and recently these have also been exploited for the development of novel drugs against trypanosomatid parasites. Nevertheless, classical anti-folate drugs targeting dihydrofolate reductase (DHFR), such as methotrexate (MTX), are only poorly effective towards Leishmania and Trypanosoma because of pteridine reductase (PTR, EC 1.5.1.33). PTR is a reduced nicotinamide adenine dinucleotide 3



phosphate

(NADPH)-dependent

short-chain

dehydrogenase/reductase

Page 4 of 31

(SDR)

unique

to

trypanosomatid parasites and mainly involved in the reduction of conjugated and unconjugated pterins, such as biopterin (Figure SI-1)17,18. Under physiological conditions PTR accounts only for the 10 % of tetrahydrofolate but, when the DHFR activity is blocked, the PTR gene is upregulated and PTR provides reduced folates necessary for parasite survival, thereby creating a metabolic bypass to DHFR inhibition19,20. PTR knockdown studies on the T. brucei bloodstream form, performed using RNA interference methodologies, proved that the parasite growth is compromised both in vitro and in the animal host20,21. Given the importance of this enzyme for parasite survival, PTR represents a promising target for the treatment of HAT. During the NMTrypI European project, various inhibitors have been developed against Trypanosoma enzymes belonging to the folate pathway. Cycloguanil (Figure 1), the active form of the pro-drug proguanil, is a known DHFR inhibitor22. Kinetic studies performed on the isolated T. brucei DHFR (TbDHFR) yielded an inhibition constant (Ki) of 256 nM towards this target, whereas a Ki of 5.8 M was associated with L. major DHFR22,23. Cycloguanil has a modest anti-parasitic effect on T. brucei (EC50 up to 25 M) but it is almost inactive on L. major promastigotes (EC50 > 500 M)22,23. It is noteworthy that the two triazine-based compounds melamine and cyromazine (Figure 1) which were previously investigated as TbPTR1 inhibitors, yielded poor activity towards this target (Ki > 35 M)24. In the present study, we provide experimental and structural evidence of cycloguanil being a TbPTR1 inhibitor. Structural information guided the design of cycloguanil derivatives based on a 1,6dihydrotriazine molecular core (Table 1). Two potent TbPTR1 inhibitors (1 and 2a), associated with IC50 values in the nano molar range, were identified. The structure of TbPTR1 was solved in complex with these two inhibitors and compared with cycloguanil to explain their improved potency. Following this, cell growth inhibition assays were performed on T. brucei and the early toxicity profiles were determined for the compounds. Considering the efficacy of cycloguanil as DHFR inhibitor against parasite diseases (e.g. malaria), the development of triazine derivatives acting as

4


Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potent dual PTR1 and DHFR inhibitors would represent a step forward in the design of new effective HAT treatments. Cl

NH2

NH2 N

N

N N

N H

NH2

Cycloguanil

NH2

N

N NH2

N

H 2N

Cyromazine

N N

NH2

Melamine

Figure 1. Chemical structures of cycloguanil, cyromazine and melamine.

RESULTS AND DISCUSSION Synthesis of 2,4-diamino-1,6-dihydrotriazine derivatives and inhibition of TbPTR1 The chemical structures of the synthesized compounds are displayed in Table 1.

Table 1. Chemical structures of cycloguanil and the synthesized 2,4-diamino-1,6-dihydro-1,3,5triazine 1 and 2a-c. Compound

Structure Cl

Cycloguanil

NH2 N

N NH2

N Cl

1

Cl

NH2 N

N NH2

N

Cl

2a

Cl

NH2 N

N NH2

N O 2N

NH2 N

2b

N N

5


NH2


Page 6 of 31

NH2 N

2c

Cl

N N

NH2

Cl

The dichloro-analogue of cycloguanil (1) was prepared by a one-step acid-catalysed cyclocondensation between equimolar 3,4-dichloroaniline and cyanoguanidine in acetone (3-component synthesis) to give directly the desired triazine as hydrochloric salt (Scheme 1), as previously described25.

NH2 N

+

Cl

H 2N

Cl

Cl

CN

NH2

a Cl

NH2

N

N N

HCl NH2

1

Scheme 1. a) 37 % w/w HCl aq., acetone, r.t., 2h.

In contrast, the 2-aryl-1,6-dihydrotriazines 2a-c were obtained through a two-component synthesis, relying on a two-steps condensation of the preformed arylbiguanide 3a-b and an aromatic carbonyl derivative, such as 3,4-dichlorobenzaldheyde (for 2a,c) or benzaldehyde (for 2b) (Scheme 2). The progress of the reaction was monitored by cuprammonium sulphate assay26.

NH2 R

N

+ H 2N

CN NH2

H N

a R

H N NH

NH2 NH

3a R= 3,4-Cl 3b R= -H

6

NH2

R


b

N R1

N N

HCl NH2

2a R= 3,4-Cl; R1= 4-NO2 2b R and R1= -H 2c R= -H; R1= 3,4-Cl

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 2. a) 37 % w/w HCl aq. (1 eq.), neat, 170 °C, 10 min. b) aryl aldehyde (1.5 eq.), 37 % w/w HCl aq. (1.1 eq.), MeOH, rifl. 2-12 h.

Activity inhibition assays identified cycloguanil as a modest inhibitor of TbPTR1, yielding an IC50 of 31.6 μM (Table 2). The four synthesized derivatives (Table 1) were preliminary investigated towards TbPTR1 through fixed-concentration inhibition assays. Compounds showing an inhibition percentage >90 % at 50 M were selected for IC50 determination and toxicity evaluation. Among the synthesized 2,4-diamino-1,6-dihydrotriazines, 1 and 2a were the most potent derivatives of this series, yielding IC50 of 692 nM and 186 nM, respectively (Table 2). However, 2b and 2c were less active (IC50 of 10.5 μM and 39.5 μM, respectively), suggesting that the introduction of halogen atoms on the phenyl moiety in position 1 improves the inhibitor potency.

Table 2. TbPTR1 inhibition, anti-parasitic activity against T. brucei, and LogP(o/w) of cycloguanil and 2,4-diamino-1,6-dihydrotriazine derivatives 1 and 2a-c. Compound

TbPTR1 inhibition IC50 in μMa

Cycloguanil 1 2a 2b 2c

31.6 0.692 0.186 10.5 39.5

T. brucei anti-parasitic activity % cell growth inhibition at 50 µM and/or cEC50 in μMa ≤ 25.0 M23 23.0 % - > 100 M 99.8 % - 29.0 M 5.00 % - > 100 M NI

LogP(o/w)27 1.61 2.02 2.26 2.03 2.92

Standard deviation is ±10 % of the value NI. No inhibition a

Overall structure and active site architecture of TbPTR1 Cycloguanil and the most active compounds 1 (IC50: 692 nM) and 2a (IC50: 186 nM) were used for structural investigations. The three complexes were characterized to resolutions ranging from 1.50 to 1.92 Å (Table SI-1). In all complexes, the crystal asymmetric unit contains a whole TbPTR1 tetramer representing the enzyme functional unit, whose structure is highly conserved to those previously

7



Page 8 of 31

described18,28–30. NADP(H), acting as cofactor for PTR enzymes, binds in an extended conformation stabilized by a tight network of conserved H-bonds18,31. Cofactor binding is essential to create both the catalytic site and the substrate binding pocket. The pterin moiety of substrates and substrate-like inhibitors bind in this pocket forming a peculiar π-sandwich between the nicotinamide ring of NADP(H) and the aromatic side chain of Phe9718,31. Within the TbPTR1 active site, substrates and inhibitors are further stabilized by the interactions with the flexible substrate binding loop (residues 207-215), which binds also the cofactor. A correlation between the occupancy of the cofactor binding pocket and the flexibility of the substrate binding loop was previously observed by us28,30. Cycloguanil and its derivatives were observed to populate the substrate binding pocket in all complexes (inhibitor and cofactor estimated occupancies are summarized in Table SI-2).

TbPTR1–NADP(H)–cycloguanil The 2,4-diamino-1,6-dihydrotriazine moiety of cycloguanil occupies the biopterin binding pocket forming a tight network of H-bonds with the cofactor and several residues having a prominent role in the catalytic process, such as Tyr174 and Ser95 (Figure 2A and SI-2A). The amino group in position 2 on the 2,4-diamino-1,6-dihydrotriazine core donates a H-bond to the Tyr174 hydroxyl that further donates a H-bond to Asp161. The nitrogen in position 3 accepts a H-bond from the ribose hydroxyl of NADP(H). Furthermore, the amine moiety in position 4, donates H-bonds to both the hydroxyl and the backbone carbonyl of Ser95. One of the methyl groups in position 6 on the 1,6-dihydrotriazine ring forms van der Waals interactions with both Phe97 and Pro210. The 4-chloro-phenyl moiety in position 1 is rotated by  80° with respect to the 1,6-dihydrotriazine ring, placing the p-chlorine in an optimal position to form a halogen bond32 with the indole aromatic system of the Trp221 sidechain (the halogen points exactly to the center of the indole five-membered ring, Figure 2A).

8


Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Active site view of TbPTR1 (white cartoon and carbon atoms, interacting residues in sticks) in complex with the cofactor NADP(H) (in sticks, grey carbons) and A) cycloguanil (in sticks, green carbons), B) 1 (in sticks, cyan carbons), and C) 2a (in sticks, purple carbons). Hydrogen and halogen 9



Page 10 of 31

bond interactions within the active site are shown as tan dashed and light blue dotted lines, respectively. In all panels, inhibitor molecules are surrounded by their omit map (displayed as green wire, contoured at the 2.5 σ level). In all panels, oxygens atoms are coloured red, nitrogen blue, phosphorous magenta, and chlorine black. Stereo-views are shown in Figure SI-2.

TbPTR1–NADP(H)–1 The binding mode adopted by 1 within the TbPTR1 catalytic pocket closely resembles that displayed by cycloguanil (showing also conserved interactions inside the cavity, Figure 2B and SI-2B). Two binding modes were observed within the cavity, resulting from two alternative orientations of the 3,4dichloro-phenyl moiety of 1. In the main orientation, the m-chlorine is accommodated inside the pocket formed by Val206, Val209, the backbone oxygen of Ser207, and the nicotinamide oxygen of the cofactor (Figure 2B). The orientation of the m-chlorine within this pocket is compatible with a weak halogen bond involving the backbone carbonyl of Ser20733 (omitted for clarity in Figure 2B). The alternative orientation of the 3,4-dichloro-phenyl moiety is visible to a partial occupancy (estimated to 30 %, Table SI-2) only in chain B. In this orientation, the 3,4-dichloro-phenyl moiety is rotated by 180° with respect to the former, directing the m-chlorine substituent towards the solvent exposed protein surface. Despite this rotation, the halogen bond between the p-chlorine and Trp221 is unaltered, supporting the prominent contribution of this interaction in inhibitor binding.

TbPTR1–NADP(H)–2a The 2,4-diamino-1,6-dihydrotriazine core of 2a adopts a pose similar to those observed for the corresponding molecular scaffolds of cycloguanil and 1, showing also unaltered interactions within the TbPTR1 active site (Figure 2C and SI-2C). In contrast to cycloguanil and 1, 2a possesses a 4nitro-phenyl substituent in position 6 on the 1,6-dihydrotriazine core (Table 1). The aromatic systems in position 1 and 6 are rotated by  73° and  48°, respectively, with respect to the dihydrotriazine

10


Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


plane. The p-chlorine on the phenyl in position 1 is directed towards the sidechains of Trp221 and Met213, without forming specific interactions with them (Figure 2C). On the other hand, the mchlorine is located in the pocket formed by Val206, Leu209, and the backbone carbonyls of Ser207 and Leu208. The orientation of the chlorine in this pocket supports the formation of two weak halogen bonds33 with the Ser207 and Leu208 backbone carbonyls (omitted for clarity in Figure 2C). In contrast to cycloguanil and 1, the accommodation of the bulkier 4-nitro-phenyl substituent of 2a induces a significant rearrangement of the substrate binding loop. This causes a shift of the 3,4dichloro-phenyl moiety toward the “floor” of the substrate binding cavity into the pocket formed by residues 206-209, allowing optimal contact interactions but increasing the distance between the pchlorine and Trp221 by about 1.0 Å. The 4-nitro-phenyl substituent in position 6 points towards the solvent exposed enzyme surface, allowing van der Waals interactions with the sidechains of Phe97 and Pro210. The orientation of the p-nitro group on this moiety is not clearly visible, indicating rotational disorder of the group (modeled only in chain D, Figure 2C; in the other subunits the nitrooxygens were excluded from our model).

Comparison of the crystal structures and SAR The comparison among the three structural complexes solved in this work showed a conserved binding mode within the TbPTR1 active site (Figure 3A). Cycloguanil and its derivatives occupied the biopterin binding pocket competing with the natural substrates (the comparison with the complex TbPTR1-folic acid24 is shown in Figure SI-3). All these compounds possess a phenyl substituent in position 1 on the 2,4-diamino-1,6-dihydrotriazine core, rotated by 73-80° with respect to the dihydrotriazine plane. The steric hindrance generated by this moiety, coupled with that of the substituents in position 6, induce the rotation of the Phe97 aromatic system which is no longer parallel to the nicotinamide of the cofactor (Figure 3B). The conserved -sandwich interaction involving the aromatic sidechain of Phe97 and the cofactor nicotinamide is essential for the substrate placement and has also a key role in inhibitor binding18,34. 11



Page 12 of 31

Figure 3. A) Active site view of the superimposition among the complexes TbPTR1–NADP(H)– cycloguanil (light green cartoon and carbon atoms; cycloguanil in sticks, green carbons), TbPTR1– NADP(H)–1 (light cyan cartoon and carbon atoms, 1 in sticks, cyan carbons), and TbPTR1– NADP(H)–2a (lilac cartoon and carbon atoms, 2a in sticks, purple carbons). The rotation of the substrate binding loop in the complex with 2a is highlighted by a red dashed arrow. B) Active site view of the superimposition between the complexes TbPTR1–NADP(H)–cycloguanil (light green cartoon and carbon atoms; cycloguanil in sticks, green carbons) and TbPTR1–NADP(H)–cyromazine (light yellow cartoon and carbon atoms; cyromazine in sticks, yellow carbons; PDB id: 2X9N35). In the complex with cycloguanil the sidechain of Phe97 is rotated by  24° with respect to the orientation 12


Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


observed in the complex with cyromazine, generating, in the former, a distortion of the conserved sandwich interaction.

The characterization of TbPTR1 in complex with the triazines melamine (Figure 1, PDB id: 3JQF24) and cyromazine (Figure 1, PDB id: 2X9N35) were previously reported showing the inhibitors sandwiched inside the biopterin binding pocket (an active site view of the complex with cyromazine is shown in Figure 3B). The comparison with cycloguanil clearly evidences that, despite the loss of aromaticity, the 2,4-diamino-1,6-dihydrotriazine core retains the same binding mode of the triazine moiety (Figure 3B). Furthermore, in the complex with cycloguanil the -sandwich is distorted by the inhibitor binding that hinders Phe97, weakening this interaction but keeping close van der Waals contacts (Figure 3B). Melamine and cyromazine have reported Ki >35 M24, indicating that they are modest TbPTR1 inhibitors. Thus, the aromaticity of the triazine core is not essential for the binding. However, H-bond interactions provides a meaningful contribution for both the 2,4-diamino-1,6dihydrotriazine of cycloguanil and the triazine of melamine and cyromazine. Conserved H-bonds are formed by their nitrogen atom in position 3 and amine moieties in positions 2 and 4 with the cofactor and several residues having a prominent role in the catalytic process (e.g. Tyr174 and Ser95). Cycloguanil and cyromazine also share hydrophobic substituents in position 6 of their triazine cores, relying on two methyl groups and a N-cyclopropyl substituent, respectively (Figure 3B). In both complexes, these hydrophobic moieties determine additional van der Waals interactions with Phe97 and residues belonging to the substrate binding loop. Nevertheless, the bulky N-cyclopropyl substituent of cyromazine induces the displacement of the substrate binding loop (Figure3B). In contrast to the triazine derivative, cycloguanil is further modified in position 1 by the introduction of a 4-chloro-phenyl moiety, which forms a halogen bond with the indole aromatic system of Trp221 (Figure 2A and 3A). The same halogen bond is also visible in the complex with 1, having a chlorine in the same position (Figure 2B and 3A). The additional m-chlorine on the phenyl moiety of 1 is accommodated in a pocket adjacent to the active site, in which it entails further halogen bonds with 13



Page 14 of 31

the substrate binding loop. These additional interactions account for its increased potency (IC50 of 692 nM) with respect to the parent compound (IC50 of 31.6 M). The same 3,4-dichloro-phenyl substituent also characterizes 2a, further having a 4-nitro-phenyl moiety in position 6 that replaces the two methyl groups of 1 and cycloguanil (Table 1). In 2a, the introduction of the bulkier 4-nitro-phenyl group induces a slightly different pose of the molecule with respect to cycloguanil and 1 (Figure 3A). Nonetheless, the interactions of the 2,4-diamino-1,6dihydrotriazine core are unaltered in all complexes (Figure 2A-C and 3A). The p-chlorine of 2a is shifted by 1.7 Å with respect to the same chlorine on 1 and cycloguanil, with subsequent loss of the halogen bond with Trp221, but gain of van der Walls contacts with the sidechain of Leu209. In addition, the m-chlorine substituent of 2a is involved in a large set of interactions that stabilizes its binding and explains the increased potency of this compound (IC50 of 186 nM) compared to 1. The placement of 2a induces a shift of the substrate binding loop, reporting a maximal displacement of 3.7 Å on Ala211 (C) with respect to the corresponding residue in the complex with cycloguanil (and 1) (Figure 3A and 4). A similar movement of the substrate binding loop is also detected in the complex with cyromazine, in which a maximal displacement of 3.4 Å on Val212 (C) is observed by the comparison with the cycloguanil complex (Figure 3B and 4). The arrangement of this loop is connected with the different set of interactions within the cavity (Figure 4A-D). In the complex with cycloguanil, two H-bonds are formed by the nicotinamide of NADP(H) and the backbone nitrogen and carbonyl of Ser207 and Leu208, respectively (Figure 4A,D). In the close proximity, Arg14 contributes to stabilize the orientation of the substrate binding loop by donating two H-bonds to the backbone carbonyls of Leu208 and Leu209. Despite the movement of the substrate binding loop observed in the complex with cyromazine the same set of interactions is formed (Figure 4B,D). On the other hand, in the complex with 2a a slightly different arrangement is observed (Figure 4C,D). The backbone carbonyl of Ser207 is rotated by 90° and accepts a H-bond from the nicotinamide nitrogen of NADP(H) (Figure 4C,D). This carbonyl is located 3.11 Å away from the m-chlorine on the 3,4-dichloro-phenyl moiety of 2a, suggesting the formation of a halogen bond with it. In the close 14


Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


proximity, Leu208 is shifted towards Arg14 that donates two H-bonds to the backbone carbonyl of the former.

Figure 4. Structural comparison among the complexes TbPTR1–NADP(H)–cycloguanil (light green cartoon and carbon atoms; cycloguanil in sticks, green carbons), TbPTR1–NADP(H)–cyromazine (light yellow cartoon and carbon atoms; cyromazine in sticks, yellow carbons; PDB id: 2X9N35), and TbPTR1–NADP(H)–2a (lilac cartoon and carbon atoms; 2a in sticks, purple carbons) A-C) Set of Hbonds (tan dashed lines) stabilizing the substrate binding loop in the complexes. The different set of interactions involving Arg14, the cofactor nicotinamide and the substrate binding loop (especially 15



Page 16 of 31

residues 207-209) is displayed. D) Superimposition among the complexes showing the different orientation adopted by the substrate binding loop. An aperture of the loop is observed in presence of both cyromazine and 2a, having bulky substituents in position 6 of the triazine and the 2,4-diamino1,6-dihydrotriazine core, respectively. A stereo-view of the D panel is shown in Figure SI-4.

Biological profile of 1, 2a-c The four synthesized 1,6-dihydrotriazines 1, 2a-c were evaluated for their in vitro anti-parasitic activity against the T. brucei bloodstream form. The anti-parasitic potential was expressed as percentage of parasitic cell growth inhibition at 50 μM (Table 2). Only 2a showed an anti-parasitic activity >80 % at 50 μM with an estimated EC50 of 29 M, whereas the other 1,6-dihydrotriazines displayed a limited efficacy towards T. brucei (cell growth inhibition 100 M). The trend of the anti-parasitic activity observed for the analyzed compounds correlates with the PTR1 inhibition data, supporting a linkage between PTR and cell growth inhibition in T. brucei. However, compound 1 shows an IC50 against PTR1 of 186 nM and an EC50 against cells of 29 µM, representing a 155-fold decrease in potency from target to parasite. Similar decreases in activity between enzyme and parasite are observed also for the other analogues. The potency decrease is a general issue in medicinal chemistry studies and for our compounds, this can be due to the difficulties in the trans-membrane passage or to off-target effects. The evaluation of the LogP values (Table 2), showed the lowest value (1.61) and the highest hydrophilicity for cycloguanil. Compounds 1, 2a-2c yielded very similar values, without an evident relationship with their anti-parasitic potency. To validate the 2,4-diamino-1,6-dihydrotriazine core as a safe scaffold for a drug discovery program, the early in vitro toxicological properties of 1, 2a-2c were assayed. The inhibitory activity towards the hERG and five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were assessed together with the mitochondrial toxicity and the cytotoxicity against human A549 cells 16


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(adenocarcinoma human alveolar basal epithelial cells) and THP-1 macrophages (data are shown in Table SI-3). All the tested compounds inhibited hERG and CYP2D6 (IC50 in the range 0.51 – 77.3 μM and 0.04 – 8.85 μM, respectively), resulting in a low Selectivity Index (SI, given by the ratio between the off-target IC50 and the TbPTR1 IC50, SIhERG/TbPTR1: 1.7 – 111; SICYP2D6/TbPTR1: 0.21 – 0.36). However, these were neither mitotoxic nor active against the other P450 CYP isoforms. Only 1 and 2a were cytotoxic against the A549 cell line with GI50 (compound concentration required for 50 % of cell growth inhibition) of 0.31 μM and 11 μM, respectively. The cytotoxicity of 1 could be due to the inhibition of hDHFR (Ki of 0.11 μM36). Furthermore, the compounds were assessed for cytotoxicity on THP-1 macrophage-like cells to determine the NOAEL (no observed adverse effect level at 50 μM) and none of them showed meaningful cytotoxicity at the tested concentration.

CONCLUSION In this work, we have exploited cycloguanil, a known inhibitor of plasmodial and trypanosomal DHFR-TS enzymes16,23,37, as a scaffold for the development of four derivatives with variable substitution in positions 1 and 6 of the 2,4-diamino-1,6-dihydrotriazine ring (Table 1). The structural characterization of TbPTR1 in complex with cycloguanil, elucidated the binding mode of this modest inhibitor and supported the rational design of the four 2,4-diamino-1,6-dihydrotriazine derivatives. Functional studies against TbPTR1 revealed that the two synthesized derivatives 1 and 2a were potent TbPTR1 inhibitors (IC50 of 692 and 186 nM, respectively). The basis of the improved potency of these inhibitors was elucidated by structural analysis that evidenced the key binging contributions provided by halogen and hydrogen bonds and by van der Waals interactions. Furthermore, the complex with 2a highlights the displacement of the substrate binding loop induced by inhibitor binging in the catalytic cavity. In addition, compound 2a was also active in blocking parasite cell growth in vitro. While enlarging the compound library for SAR studies, we will pay attention to the mechanism of parasite internalization and substituents/fragments will be introduced to modulate their lipophilicity. Although the early in vitro toxicological studies indicated a few liabilities for 1 and 2a, 17



Page 18 of 31

all of which should be overcome by SAR studies, our data provides new important insights for the development of 2,4-diamino-1,6-dihydrotriazine derivatives as potent TbPTR1 inhibitors. Giving the effectiveness of cycloguanil on DHFR enzymes, 2,4-diamino-1,6-dihydrotriazine derivatives represent a new resource exploitable for the development of dual PTR1 and DHFR inhibitors with anti-parasitic activity. Additionally, the new compounds can be explored for their capacity to inhibit PTR enzymes from other organisms, such as PTR1 from Leishmania spp and from Trypanosoma cruzi. The similarity of the target structure among the different organisms suggest that the triazine core can be developed further for a pan-PTR1 enzymes inhibition.

EXPERIMENTAL SECTION Synthetic procedures All commercially available chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. Reactions were monitored by thin-layer chromatography on silica gel plates (60F-254, E. Merck) and visualized with UV light, iodine vapours, cerium ammonium sulphate or alkaline KMnO4 aqueous solution. The following solvents and reagents have been abbreviated: ethyl ether (Et2O), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (DCM), methanol (MeOH). All reactions were carried out with standard techniques or under microwave irradiation. NMR spectra were recorded on a Bruker 400 spectrometer with 1H at 400.134 MHz and

13C

at 100.62 MHz. Proton chemical shift was referenced to the residual solvent peak.

Chemical shifts are reported in parts per million (ppm, δ units). Coupling constants are reported in units of Hertz (Hz). Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q quartet; dd, double doublet; m, multiplet; b, broad. Mass spectra were obtained on a 6520 Accurate-Mass Q-TOF LC/MS and 6310A Ion TrapLC-MS(n). The melting point was recorded on a Stuart, SMP3 (Barloworld Scientific Limited Stone, Staffordshire, UK) and was uncorrected. (a) 1-(3,4-dichlorophenyl)-6,6-dimethyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (1) 18


Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To a solution of 3,4-dichloroaniline (1 eq, 162 mg, 1 mmol) and N-cyano-guanidine (1 eq., 84 mg, 1 mmol) in acetone (15 ml) concentrated hydrochloric acid 37 % w/w (1 eq., 83 µL, 1 mmol) was added dropwise. The resulting suspension was stirred at room temperature for 5 hours. The precipitate was collected by filtration and washed with cold acetone to give the desired product as hydrochloric salt without further purification needed. White crystalline solid (71 % yield). 1HNMR (400 MHz, DMSOd6) δ: 1.37 (s, 6H, CH3); 6.37 (bs, 2H, NH2); 6.66 (bs, 1H, NH); 7.43 (dd, 1H, J = 4.0, 8.0 Hz, CHar); 7.78-7.80 (m, 3H, CHar, NH2); 9.30 (s, 1H, CHar). 13CNMR (101 MHz, DMSO-d6) δ: 27.15; 27.24; 69.90; 130.61; 131.86; 132.35; 132.39; 132.80; 134.73; 156.82; 157.39. m.p. [> 200 °C]. HRMS m/z [M+H]+ Calcd for C11H14[35Cl]2N5: 286.0621, found: 286.0622; calcd for C11H14[35Cl][37Cl]N5: 288.0591, found: 288.0590. (b) 1-(3,4-dichlorophenyl)-6-(4-nitrophenyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine (2a) Neat 3,4-dichlroaniline (1 eq., 162 mg, 1 mmol), N-cyano-guanidine (1 eq., 84 mg, 1 mmol) and concentrated hydrochloric acid 37 % w/w (1 eq., 83 μL, 1 mmol) were gradually heated at 170 °C and reacted for 10 minutes. The solid formed was chilled down, triturated over acetone and filtered to give the N1-(3,4-dichlorophenyl)-diguanide hydrochloride (3a) used in the next step without further purification. 3a (1 eq., 167 mg, 0.60 mmol) was suspended in MeOH (15 mL) and 4-nitrobenzaldehyde (1.5 eq., 136 mg, 0.90 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq., 58 µL, 0.7 mmol) were added. The mixture was refluxed for 12 hours. Thereafter, the mixture was neutralized with Na2CO3 and the solvent evaporated. The crude was directly purified on silica gel (eluent DCM:MeOH 9:1) obtaining a brown solid (38 % yield). 1H NMR (400 MHz, Methanol-d4) δ 6.29 (s, 1H), 7.18 (dd, J = 2.5, 8.6 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 71.37, 125.37, 129.39, 130.02, 131.59, 133.34, 134.94, 134.98, 138.12, 145.56, 150.27, 159.05, 159.22. m.p. [dec. 185 °C]. HRMS m/z [M+H]+ Calcd for C15H12[35Cl]2N6O2: 379.0472, found: 379.0469; calcd for C15H12[35Cl] [37Cl]N6O2: 381.0442, found: 381.0440. (c) 1,6-diphenyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (2b) 19



Page 20 of 31

Neat aniline (1 eq., 452 µL, 5 mmol), N-cyano-guanidine (1.1 eq., 462 mg, 5.5 mmol) and concentrated hydrochloric acid 37 % w/w (1 eq., 500 µL, 1 mmol) were gradually heated at 170 °C and reacted for 10 minutes. The solid formed was chilled down, triturated over acetone and filtered to give the N1-phenyl-diguanide hydrochloride (3b) as a pink solid used in the next step without further purification. 3b (1 eq., 500 mg, 2.20 mmol) was suspended in MeOH (25 mL) and benzaldehyde (1.5 eq., 290 µL, 3.30 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq., 1.67 mL 2.10 mmol) were added. The mixture was refluxed for 4 hours and concentrated. The crude was suspended in water and washed with AcOEt. The aqueous phase was concentrated and the residue triturated over Et2O to give a white crystalline solid (52 % yield). 1HNMR (400 MHz, DMSO-d6) δ: 5.28 (bs, 2H, NH2), 6.08 (s, 1H, CHtriazine), 7.15 (d, 2H, CHar), 7.31-7.44 (m, 10H, CHar), 7.70 (s, 2H, NH2), 9.37 (bs, 1H, NH). 13CNMR (101 MHz, DMSO-d6) δ: 69.88, 121.38, 123.31, 126.80, 127.37, 128.54, 128.76, 128.80, 129.32, 129.60, 129.95, 137.92, 138.72, 157.05, 157.26. m.p. [> 200 °C]. HRMS m/z [M+H]+ Calcd for C15H16N5: 266.3275, found: 266.3272. (d) 6-(3,4-dichlorophenyl)-1-phenyl-1,6-dihydro-1,3,5-triazine-2,4-diamine hydrochloride (2c) 3b (1 eq., 28 mg, 0.15 mmol) was suspended in MeOH (5 mL) and 3,4-dichlorobenzaldehyde (1.5 eq., 40 mg, 0.23 mmol) and concentrated hydrochloric acid 37 % w/w (1.1 eq., 0.21 mL, 0.17 mmol) were added. The mixture was refluxed for 24 hours and concentrated. The crude was triturated over EtOH and the precipitate obtained crystalized from Et2O to give 35 mg of a white crystalline solid (69 % yield). 1H NMR (400 MHz, DMSO-d6) δ 6.43 (s, 1H, CHtriazine), 7.03 - 7.07 (m, 3H, CHar, NH), 7.24 - 7.34 (m, 7H, CHar, NH), 7.34 - 7.40 (m, 2H, CHar), 9.71 (s, 1H, NH). m.p. [> 200 °C]. HRMS m/z [M+H]+ Calcd for C15H14[35Cl]2N5: 334.0621. Found: 334.0621; calcd for C15H14[35Cl] [37Cl]N5: 336.0591, found: 336.0593.

TbPTR1 expression, purification, and enzymatic assays Recombinant TbPTR1 was expressed and purified by established methods28. TbPTR1 enzyme activity was measured using the in vitro coupled assay reported by Shanks et al.38. The assay follows 20


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the reduction of H2B to H4B by PTR enzyme. H4B is further oxidized to qH2B by cytochrome c Fe3+, whose reduction to cytochrome c Fe2+ is spectrophotometrically monitored at 550 nm. The IC50 values determined for the compounds are summarized in Table 2.

Protein crystallization Crystallization was performed by the vapor diffusion hanging drop technique at room temperature39. Well-ordered monoclinic crystals of histidine tagged TbPTR1 were grown within few days using a precipitant solution composed of 2-2.5 M sodium acetate and 0.1 M sodium citrate, pH 5, as described elsewhere29. The ternary complexes TbPTR1–cofactor–inhibitor were obtained by the soaking technique. Compounds, solubilized in DMSO (50 mM), were added to the cryo-protectant solution (prepared by adding 30 % v/v glycerol to the precipitant) to reach final concentrations of 4-5 mM (without exceeding a 10 % v/v DMSO concentration). Preformed protein crystals were then transferred to the cryo-protectant/soaking solution, and flash-frozen in liquid nitrogen after 24-48 hours.

Data collection, structure solution and refinement Diffraction data were collected using the synchrotron radiation at the Diamond Light Source (DLS, Didcot, United Kingdom) beamline I02 and at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) beamline ID30-B, both equipped with a Dectris Pilatus 6M-F detector. Diffraction data were integrated using Mosflm40 and scaled with Scala41 from the CCP4 suite42. Data collection statistics are reported in Table SI-1. Crystals of TbPTR1 belonged to the primitive monoclinic space group P21, with a functional tetramer in the asymmetric unit. Structures were solved by the molecular replacement technique, as implemented in the software MOLREP43. A functional tetramer of TbPTR1 (PDB id 5JDC28) was used as searching model (solvent and non-protein molecules were preventively excluded). All models were refined using REFMAC544 from the CCP4 suite. The molecular graphic software Coot45,46 was used for visual inspection and manual rebuilding of missing atoms in the 21



Page 22 of 31

protein models. The occupancy of the exogenous ligands was adjusted and refined to a value resulting in atomic displacement parameters close to those of neighboring protein atoms in fully occupied sites. Water molecules were added using the ARP/wARP47 suite and checked with Coot. The final models were inspected manually and checked with the programs Coot and Procheck48. Data refinement statistics are reported in Table SI-1. The structural models were rendered using CCP4mg49. Final coordinates and structure factors were deposited in the Protein Data Bank under the codes 6HNC (TbPTR1–NADP(H)–cycloguanil), 6HNR (TbPTR1–NADP(H)–1), and 6HOW (TbPTR1– NADP(H)–2a).

Evaluation of activity against T. brucei Anti-trypanosomal activity tests were performed as described elsewhere50. Briefly, T. brucei Lister 427 bloodstream forms were incubated in the presence of compounds for 72 h, followed by cell lysis and addition of the Sybr Green I dye (Life Technologies). The plates were then read for fluorescence at the microplate reader Envision (PerkinElmer). Compounds that showed percentage of inhibition of parasite growth higher than 80 % at 50 M were selected for EC50 determination.

Determination of in vitro toxicological properties The determination of the early toxicity profile of 1, 2a-c, comprising the hERG, cytochrome P450 (1A2, 2C9, 2C19, 2D6 and 3A4), mitochondrial toxicity, cytotoxicity were preformed as previously described51. The cytotoxicity of THP-1-derived macrophages was assessed by the colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)28.

ASSOCIATED CONTENT Supporting Information contains additional figures, showing the PTR catalytic mechanism and additional stereo-views of the complexes, and tables, including data collection, structure solution and 22


Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


refinement statistics, inhibitor and cofactor occupancies within the catalytic cavity, and the liability profile for the synthesized compounds.

AUTHOR INFORMATION

Corresponding authors *(C.P.) Phone: +39 0577232132. E-mail: [email protected] *(S.M.) Phone: +39 0577234255. E-mail: [email protected] Author Contributions P.L. designed/synthesized the molecules and performed the medicinal chemistry. G.W. carried out the in vitro enzyme assays. M.K. carried out the in vitro toxicological assays. C.P.B., C.B.M. carried out the in vitro antiparasitic tests and the in vitro toxicological profiles. G.L. and C.P. prepared the TbPTR1 protein and carried out the X-ray crystallographic studies. All authors analyzed the data and C.P., P.L., C.B., A.C.D.S., S.G. M.P.C. and S.M. wrote the manuscript. Notes The authors declare no competing financial interest. Authors will release the atomic coordinates and experimental data upon article publication.

ACKNOWLEDGEMENT This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI - New Medicine for Trypanosomatidic Infections). The authors acknowledge the COST Action CM1307, http://www.cost.eu/COST_Actions/cmst/CM1307 for the contribution to the discussion of the research results.

23



Page 24 of 31

The authors would also like to acknowledge Diamond Light Source (proposal MX11690) and the European Synchrotron Radian Facility for provisioning of beamtime, and in particular the staff of the DLS beamline I02 and the ESRF beamline ID30-B. We would also like to thank all the staff of the synchrotron facilities for assistance in using the beamlines. The research leading to these results received founding from the European Community Seventh Framework Programme (FP7/2007-2013) under Biostruct-X (Grant Agreement 283570). We would also like to thank the financial support provided by the MIUR Grant Dipartimento di Eccellenza 2018-2022.

ABBREVIATIONS PDB, Protein Data Bank; H2B, dihydrobiopterin; H4B, tetrahydrobiopterin; qH2B, quinonoid dihydrobiopterin; Et2O, ethyl ether; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; DCM, dichloromethane; MeOH, methanol; hERG, human ether-ago-go-related gene; THP1, human monocytic cell line; A549, human lung adenocarcinoma epithelial cell line; ATP, adenosine triphosphate; DMEM, Dulbecco's Modified Eagle Medium; FCS, fetal calf serum

REFERENCES (1) Castillo, E., Dea-Ayuela, M. A., Bolás-Fernández, F., Rangel, M., and González-Rosende, M. E. (2010) The kinetoplastid chemotherapy revisited: current drugs, recent advances and future perspectives. Curr. Med. Chem. 17, 4027–4051. (2) Machado-Silva, A., Guimarães, P. P. G., Tavares, C. A. P., and Sinisterra, R. D. (2015) New perspectives for leishmaniasis chemotherapy over current anti-leishmanial drugs: a patent landscape. Expert Opin. Ther. Pat. 25, 247–260. (3) Delespaux, V., and de Koning, H. P. (2007) Drugs and drug resistance in African trypanosomiasis. Drug Resist. Updat. 10, 30–50. (4) Baker, N., de Koning, H. P., Mäser, P., and Horn, D. (2013) Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 29, 110–118. 24


Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) Alirol, E., Schrumpf, D., Amici Heradi, J., Riedel, A., de Patoul, C., Quere, M., and Chappuis, F. (2013) Nifurtimox-Eflornithine Combination Therapy for Second-Stage Gambiense Human African Trypanosomiasis: Médecins Sans Frontières Experience in the Democratic Republic of the Congo. Clin. Infect. Dis. 56, 195–203. (6) Torreele, E., Trunz, B. B., Tweats, D., Kaiser, M., Brun, R., Mazué, G., Bray, M. A., and Pécoul, B. (2010) Fexinidazole – A New Oral Nitroimidazole Drug Candidate Entering Clinical Development for the Treatment of Sleeping Sickness. PLoS Negl. Trop. Dis. 4, e923. (7) Maxmen, A. (2017) Sleeping sickness can now be cured with pills. Nat. News 550, 441. (8) Dawidowski, M., Emmanouilidis, L., Kalel, V. C., Tripsianes, K., Schorpp, K., Hadian, K., Kaiser, M., Mäser, P., Kolonko, M., Tanghe, S., Rodriguez, A., Schliebs, W., Erdmann, R., Sattler, M., and Popowicz, G. M. (2017) Inhibitors of PEX14 disrupt protein import into glycosomes and kill Trypanosoma parasites. Science 355, 1416–1420. (9) Fairlamb, A. H. (2003) Chemotherapy of human African trypanosomiasis: current and future prospects. Trends Parasitol. 19, 488–494. (10) Jacobs, R. T., Nare, B., and Phillips, M. A. (2011) State of the Art in African Trypanosome Drug Discovery. Curr. Top. Med. Chem. 11, 1255–1274. (11) Steverding, D., Sexton, D. W., Wang, X., Gehrke, S. S., Wagner, G. K., and Caffrey, C. R. (2012) Trypanosoma brucei: Chemical evidence that cathepsin L is essential for survival and a relevant drug target. Int. J. Parasitol. 42, 481–488. (12) Martyn, D. C., Jones, D. C., Fairlamb, A. H., and Clardy, J. (2007) High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity. Bioorg. Med. Chem. Lett. 17, 1280–1283. (13) Bowyer, P. W., Tate, E. W., Leatherbarrow, R. J., Holder, A. A., Smith, D. F., and Brown, K. A. (2008) N-Myristoyltransferase: a Prospective Drug Target for Protozoan Parasites. ChemMedChem 3, 402–408. (14) Voorhis, W. C. V., Rivas, K. L., Bendale, P., Nallan, L., Hornéy, C., Barrett, L. K., Bauer, K. 25



Page 26 of 31

D., Smart, B. P., Ankala, S., Hucke, O., Verlinde, C. L. M. J., Chakrabarti, D., Strickland, C., Yokoyama, K., Buckner, F. S., Hamilton, A. D., Williams, D. K., Lombardo, L. J., Floyd, D., and Gelb, M. H. (2007) Efficacy, Pharmacokinetics, and Metabolism of Tetrahydroquinoline Inhibitors of Plasmodium falciparum Protein Farnesyltransferase. Antimicrob. Agents Chemother. 51, 3659– 3671. (15) Hawser, S., Lociuro, S., and Islam, K. (2006) Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol. 71, 941–948. (16) Yuthavong, Y., Yuvaniyama, J., Chitnumsub, P., Vanichtanankul, J., Chusacultanachai, S., Tarnchompoo, B., Vilaivan, T., and Kamchonwongpaisan, S. (2005) Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors. Parasitology 130, 249–259. (17) Vickers, T. J., and Beverley, S. M. (2011) Folate metabolic pathways in Leishmania. Essays Biochem. 51, 63–80. (18) Dawson, A., Gibellini, F., Sienkiewicz, N., Tulloch, L. B., Fyfe, P. K., McLuskey, K., Fairlamb, A. H., and Hunter, W. N. (2006) Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate. Mol. Microbiol. 61, 1457–1468. (19) Bello, A. R., Nare, B., Freedman, D., Hardy, L., and Beverley, S. M. (1994) PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc. Natl. Acad. Sci. U. S. A. 91, 11442–11446. (20) Sienkiewicz, N., Ong, H. B., and Fairlamb, A. H. (2010) Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice. Mol. Microbiol. 77, 658–671. (21) Ong, H. B., Sienkiewicz, N., Wyllie, S., and Fairlamb, A. H. (2011) Dissecting the Metabolic Roles of Pteridine Reductase 1 in Trypanosoma brucei and Leishmania major. J. Biol. Chem. 286, 10429–10438. (22) Gilbert, I. H. (2002) Inhibitors of dihydrofolate reductase in leishmania and trypanosomes. Biochim. Biophys. Acta BBA - Mol. Basis Dis. 1587, 249–257. 26


Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Vanichtanankul, J., Taweechai, S., Yuvaniyama, J., Vilaivan, T., Chitnumsub, P., Kamchonwongpaisan, S., and Yuthavong, Y. (2011) Trypanosomal Dihydrofolate Reductase Reveals Natural Antifolate Resistance. ACS Chem. Biol. 6, 905–911. (24) Tulloch, L. B., Martini, V. P., Iulek, J., Huggan, J. K., Lee, J. H., Gibson, C. L., Smith, T. K., Suckling, C. J., and Hunter, W. N. (2010) Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the Leishmaniases. J. Med. Chem. 53, 221–229. (25) Modest, E. J. (1956) Chemical and Biological Studies on 1,2-Dihydro-s-triazines. II. ThreeComponent Synthesis. J. Org. Chem. 21, 1–13. (26) Modest, E. J., and Levine, P. (1956) Chemical and Biological Studies on 1,2-Dihydro-s-triazines. III. Two-Component Synthesis. J. Org. Chem. 21, 14–20. (27) Schrödinger, L. (2017) Schrödinger Release 2017-1: QikProp. New York, NY. (28) Borsari, C., Luciani, R., Pozzi, C., Poehner, I., Henrich, S., Trande, M., Cordeiro-da-Silva, A., Santarem, N., Baptista, C., Tait, A., Di Pisa, F., Dello Iacono, L., Landi, G., Gul, S., Wolf, M., Kuzikov, M., Ellinger, B., Reinshagen, J., Witt, G., Gribbon, P., Kohler, M., Keminer, O., Behrens, B., Costantino, L., Tejera Nevado, P., Bifeld, E., Eick, J., Clos, J., Torrado, J., Jiménez-Antón, M. D., Corral, M. J., Alunda, J. M., Pellati, F., Wade, R. C., Ferrari, S., Mangani, S., and Costi, M. P. (2016) Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs. J. Med. Chem. 59, 7598–7616. (29) Linciano, P., Dawson, A., Pöhner, I., Costa, D. M., Sá, M. S., Cordeiro-da-Silva, A., Luciani, R., Gul, S., Witt, G., Ellinger, B., Kuzikov, M., Gribbon, P., Reinshagen, J., Wolf, M., Behrens, B., Hannaert, V., Michels, P. A. M., Nerini, E., Pozzi, C., di Pisa, F., Landi, G., Santarem, N., Ferrari, S., Saxena, P., Lazzari, S., Cannazza, G., Freitas-Junior, L. H., Moraes, C. B., Pascoalino, B. S., Alcântara, L. M., Bertolacini, C. P., Fontana, V., Wittig, U., Müller, W., Wade, R. C., Hunter, W. N., Mangani, S., Costantino, L., and Costi, M. P. (2017) Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery. ACS Omega 2, 5666–5683. 27



Page 28 of 31

(30) Di Pisa, F., Landi, G., Dello Iacono, L., Pozzi, C., Borsari, C., Ferrari, S., Santucci, M., Santarem, N., Cordeiro-da-Silva, A., Moraes, C. B., Alcantara, L. M., Fontana, V., Freitas-Junior, L. H., Gul, S., Kuzikov, M., Behrens, B., Pöhner, I., Wade, R. C., Costi, M. P., and Mangani, S. (2017) Chroman4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity. Molecules 22, 426. (31) Gourley, D. G., Schüttelkopf, A. W., Leonard, G. A., Luba, J., Hardy, L. W., Beverley, S. M., and Hunter, W. N. (2001) Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites. Nat. Struct. Biol. 8, 521–525. (32) Cavallo, G., Metrangolo, P., Milani, R., Pilati, T., Priimagi, A., Resnati, G., and Terraneo, G. (2016) The Halogen Bond. Chem. Rev. 116, 2478–2601. (33) Wilcken, R., Zimmermann, M. O., Lange, A., Joerger, A. C., and Boeckler, F. M. (2013) Principles and Applications of Halogen Bonding in Medicinal Chemistry and Chemical Biology. J. Med. Chem. 56, 1363–1388. (34) Pozzi, C., Tassone, G., and Mangani, S. (2018) Chapter Five - X-ray Crystallography Contributions to Drug Discovery Against Parasite, in Annual Reports in Medicinal Chemistry (Botta, M., Ed.), pp 175–230. Academic Press. (35) Dawson, A., Tulloch, L. B., Barrack, K. L., and Hunter, W. N. (2010) High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target. Acta Crystallogr. D Biol. Crystallogr. 66, 1334–1340. (36) Tonelli, M., Naesens, L., Gazzarrini, S., Santucci, M., Cichero, E., Tasso, B., Moroni, A., Costi, M. P., and Loddo, R. (2017) Host dihydrofolate reductase (DHFR)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus. Eur. J. Med. Chem. 135, 467–478. (37) Chitnumsub, P., Yuvaniyama, J., Chahomchuen, T., Vilaivan, T., and Yuthavong, Y. (2009) Crystallization and preliminary crystallographic studies of dihydrofolate reductase-thymidylate 28


Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


synthase from Trypanosoma cruzi, the Chagas disease pathogen. Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 65, 1175–1178. (38) Shanks, E. J., Ong, H. B., Robinson, D. A., Thompson, S., Sienkiewicz, N., Fairlamb, A. H., and Frearson, J. A. (2010) Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase. Anal. Biochem. 396, 194–203. (39) Benvenuti, M., and Mangani, S. (2007) Crystallization of soluble proteins in vapor diffusion for x-ray crystallography. Nat. Protoc. 2, 1633–1651. (40) Powell, H. R., Johnson, O., and Leslie, A. G. W. (2013) Autoindexing diffraction images with iMosflm. Acta Crystallogr. D Biol. Crystallogr. 69, 1195–1203. (41) Evans, P. (2006) Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82. (42) Collaborative Computational Project, Number 4. (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763. (43) Vagin, A., and Teplyakov, A. (2010) Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25. (44) Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367. (45) Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. (46) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501. (47) Langer, G., Cohen, S. X., Lamzin, V. S., and Perrakis, A. (2008) Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179. (48) Laskowski, R. A., MacArthur, M. W., and Thornton, J. M. (1998) Validation of protein models derived from experiment. Curr. Opin. Struct. Biol. 8, 631–639. 29



Page 30 of 31

(49) Potterton, L., McNicholas, S., Krissinel, E., Gruber, J., Cowtan, K., Emsley, P., Murshudov, G. N., Cohen, S., Perrakis, A., and Noble, M. (2004) Developments in the CCP4 molecular-graphics project. Acta Crystallogr. D Biol. Crystallogr. 60, 2288–2294. (50) Faria, J., Moraes, C. B., Song, R., Pascoalino, B. S., Lee, N., Siqueira-Neto, J. L., Cruz, D. J. M., Parkinson, T., Ioset, J.-R., Cordeiro-da-Silva, A., and Freitas-Junior, L. H. (2015) Drug discovery for human African trypanosomiasis: identification of novel scaffolds by the newly developed HTS SYBR Green assay for Trypanosoma brucei. J. Biomol. Screen. 20, 70–81. (51) Moraes, C. B., Witt, G., Kuzikov, M., Ellinger, B., Calogeropoulou, T., Prousis, K. C., Mangani, S., Di Pisa, F., Landi, G., Iacono, L. D., Pozzi, C., Freitas-Junior, L. H., dos Santos Pascoalino, B., Bertolacini, C. P., Behrens, B., Keminer, O., Leu, J., Wolf, M., Reinshagen, J., Cordeiro-da-Silva, A., Santarem, N., Venturelli, A., Wrigley, S., Karunakaran, D., Kebede, B., Pöhner, I., Müller, W., Panecka-Hofman, J., Wade, R. C., Fenske, M., Clos, J., Alunda, J. M., Corral, M. J., Uliassi, E., Bolognesi, M. L., Linciano, P., Quotadamo, A., Ferrari, S., Santucci, M., Borsari, C., Costi, M. P., and Gul, S. (2019) Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform. SLAS Discov. Adv. Life Sci. RD 24, 346–361.

30


Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only.

31


Structural Insights into the Development of Cycloguanil Derivatives as

Recommend Documents