19 research outputs found
Design and synthesis of novel enzyme inhibitors as potential antiparasitic agents
No full textBackground
Protozoan parasitic diseases, such as Malaria and Human African Trypanosomiasis (HAT), have a tremendous health, social and economic impact on people living in tropical and subtropical regions of the world. While around 3.8 billion people are at risk of infection, available treatments are unsatisfactory, mainly due to constantly increasing drug resistance. Hence, there is an urgent need of developing new chemotherapeutic agents acting on new molecular targets, such as essential parasitic enzymes.
Aims
The aims of the present project are:
1) Synthesis of competitive inhibitors of Trypanosoma brucei isoform of N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (TbFolD), a crucial enzyme involved in the folate pathway.
2-3) Synthesis of covalent inhibitors of reactive cysteine-containing enzymes, namely Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) and rhodesain (TbCatL), which are central in energy metabolism and virulence, respectively. The covalent inhibitors designed in the present project are characterized by the 3-bromo isoxazoline nucleus as an innovative electrophilic warhead.
Methods
1) Synthesis: a) 1,3-dipolar cycloaddition reactions of bromo-nitrile oxide with suitable alkenes; b) condensation/cyclization reactions; c) coupling reactions; d) amino acid protecting groups;
2) Purification, structure assignment and purity assessment: a) flash chromatography; b) crystallization; c) 1H and 13C-NMR; d) HPLC and chiral HPLC; e) determination of chiroptical properties; f) MS analysis;
3) Biochemical and pharmacological assays: a) enzyme inhibition assays; b) identification of the binding site for covalent inhibitors through MALDI/TOF analysis; c) in vitro activity against parasites and human cell lines.
Results
1) TbFolD inhibitors(1)
We started synthesizing LY374571 (1), a known human FolD inhibitor, with the aim of using it as a lead compound: interestingly, following the published synthesis protocol we unambiguously obtained compound 2, with a structure different from the one reported in the literature. In order to understand the structural requirements for an improved activity, we performed a structure-activity relationship study replacing the glutamate tail with other α-, β- and γ-amino acids (compounds 4a-g). Compound 2 showed a Ki of 1.1 μM toward TbFolD, in the same range of 3 (Ki=8.5 μM), another human known FolD inhibitor; the affinity increased two times in the case of 4b and 4d (Ki=0.48 and 0.54 μM) while the others showed similar or higher Ki values. When assayed in vitro on the bloodstream form of T. brucei, unfortunately, only 2 showed a modest antiparasitic activity (IC50=49 μM), comparable to the one of 3 (IC50=57 μM), but with a higher selectivity index (4 versus 1.8) when assayed on THP1 monocytes. In collaboration with Prof. W. Hunter, we were able to get the first X-ray structure of TbFolD in presence of 2 and NADP+, which allowed us to rationalize the different range of affinity of the synthesized compounds and will guide the design of new inhibitors in the future.
2) PfGAPDH inhibitors(2)
The catalytic cysteine residue of GAPDH is inhibited by a number of compounds that alkylate thiols. We previously demonstrated that the 3-Br-isoxazoline nucleus is able to react with the catalytic Cys of cytidine triphosphate synthetase (CTPS), a glutamine amidotransferase. I herein evaluated the strength of this warhead for the irreversible inactivation of PfGAPDH by testing 3-Br-acivicin and a series of potential inhibitors of general structure 5, with distinct substitution patterns at the 5 position to modulate the reactivity (Figure 2).
The compounds are able to inhibit PfGAPDH with a biphasic fashion through selective covalent binding to the catalytic Cys residue. The differences in activity of the tested inhibitors were rationalized on the basis of the electron density maps and covalent docking studies.
Interestingly, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human orthologue was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer, thus evidencing an interesting selectivity profile(3).
The new inhibitors were also tested for their antiparasitic activity against chloroquine-sensitive and chloroquine resistant P. falciparum strains and some of them showed a promising activity.
3) TbCatL inhibitors(4)
A further application of the 3-bromoisoxazoline warhead in the design of covalent enzymatic inhibitors is represented by the development of new TbCatL inhibitors obtained by coupling this warhead to a proper peptide-like recognition moiety.(4) Some low micromolar inhibitors were identified, among which 6a,b and 7 showed also an interesting antiparasitic activity.
Conclusions
We described new inhibitors of parasitic enzymes using both a non-covalent (for TbFolD) and covalent (for PfGAPDH and TbCatL) approach. Covalent inhibitors were designed exploiting the reactivity of the 3-Br-isoxazoline nucleus and properly tuning the recognition moiety to confer affinity and selectivity for the selected target enzymes. The biological results were rationalized through a careful analysis of enzyme-cofactor-inhibitor co-crystal structure or covalent docking studies. These data represent a strong basis for further developments of new, more effective, antiparasitic agents.
References
(1)Eadsforth, T. C.; Pinto, A.; Luciani, R.; Tamborini, L.; Cullia, G.; De Micheli, C.; Marinelli, L.; Cosconati, S.; Novellino, E.; Lo Presti, L.; Cordeiro da Silva, A.; Conti, P.; Hunter, W. N.; Costi, M. P. Characterization of 2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl ureido based inhibitors of Trypanosoma brucei FolD and testing for antiparasitic activity. J. Med. Chem. 2015, 58(20), 7938-7948.
(2)Bruno, S.; Pinto, A.; Paredi, G.; Tamborini, L.; De Micheli, C.; La Petra, V.; Marinelli, L.; Novellino, E.; Conti, P.; Mozzarelli, A. Discovery of covalent inhibitors of glyceraldehyde 3-phosphate dehydrogenase, a target for the treatment of malaria. J. Med. Chem. 2014, 57(17), 7465–7471.
(3)Bruno, S.; Margiotta, M.; Pinto, A.; Cullia, G.; Conti, P.; De Micheli, C.; Mozzarelli, A. Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenases. Bioorg. Med. Chem. 2016, in press, doi: 10.1016/j.bmc.2016.04.033.
(4)Ettari, R.; Pinto, A.; Previti, S.; Tamborini, L.; Angelo, I. C.; La Pietra, V.; Marinelli, L.; Novellino, E.; Schirmeister, T.; Zappalà, M.; Grasso, S.; De Micheli, C.; Conti, P. Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation. Bioorg. Med. Chem. 2015, 23, 7053-7060
ENZYME INHIBITORS AS POTENTIAL ANTIPARASITIC AGENTS
No full textAbstract of the PhD thesis
ENZYME INHIBITORS AS POTENTIAL ANTIPARASITIC AGENTS
PhD course in Chemistry, XXIX cycle, University of Milan
PhD student: Gregorio Cullia (R10447)
Tutor: Prof. Paola Conti
Co-tutor: Dr. Lucia Tamborini
Protozoan parasitic diseases, such as Malaria and Human African Trypanosomiasis (HAT), have a tremendous health, social and economic impact on people living in tropical and subtropical regions of the world. While around 3.8 billion people are at risk of infection, [1] available treatments are unsatisfactory, mainly due to constantly increasing drug resistance. Hence, there is an urgent need of developing new chemotherapeutic agents acting on new molecular targets, such as essential parasitic enzymes. One selected target is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of P. falciparum, a key glycolytic enzyme with many other important functions. In this thesis, I describe the development of inhibitors that act alkylating, in a selective way, the catalytic cysteine, inspired by (S,S)-3-bromoacivicin [(S,S)-1]. [2]Some of the inhibitors, in particular (S,S)-9 and (S,S)-59, showed sub-micromolar EC50 toward P. falciparum cultures and a considerably low cytotoxicity toward human cell lines (≥ 20 mM).
In the second project, the interesting 3-bromoisoxazoline warhead was coupled to known peptidic recognition moieties generating new inhibitors of rhodesain(TbCatL), an essential protease involved in virulence and defence processes of the parasite. [3] Some low micromolar inhibitors were identified, among which the diastereoisomeric mixture (S,S,S)-138/(S,R,R)-139 showed also an interesting antiparasitic activity.
The third enzyme that was exploited as target is the N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (TbFolD), a crucial enzyme involved in the folate pathway. I started synthesizing LY374571 [(S)-149], a known human FolD inhibitor, [4] with the aim of using it as a lead compound: interestingly, following the published synthesis protocol I unambiguously obtained compound (S)-156, with a structure different from the one reported in the literature. I performed a structure-activity relationships study replacing the glutamate tail with other α-, β- and γ-amino acids [compounds (S)-175-181]. Moreover, the first X-ray structure of TbFolD in presence of (S)-156 and NADP+,was obtained. [5]
The last part of this PhD thesis regards the development of new potential agents for the treatment of C. trachomatis infections. This bacterium is the agent trachoma (a neglected tropical disease), an eye infection that causes the impairment of the sight of 1.9 million people, and of one of the most prevalent sexually transmitted infection. [6] In the past, the inhibitory activity of CI-976 (182) of inhibiting human lysophosphatidicacid:acyl-CoA acyl transferase(hLPAAT) was correlated to a delay in the slow recycling pathway of transferrin, that hampered the growth of the bacterium. [7] I performed an exhaustive structure-activity relationships study focusing on the different structural features of 182 (such as the α- and the N- substitutions and the properties of the aromatic ring). Among the synthesized compounds, 185 showed anti-chlamydial activity comparable to 182. This approach represents an innovative strategy to treat microbial infections, based on the inhibition of a trafficking pathway that is disposable for the human host.
References
[1] a. [Online]. Available: http://www.who.int/mediacentre/factsheets/fs094/en/;b. [Online]. Available: http://www.who.int/mediacentre/factsheets/fs259/en/.
[2] S. Bruno, A. Pinto, G. Paredi, L. Tamborini, C. De Micheli, V. La Pietra, L. Marinelli, E. Novellino, P. Conti and A. Mozzarelli, “Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria,” J. Med. Chem., vol. 57, pp. 7465-7471, 2014.
[3] a. R. Ettari, S. Previti, L. Tamborini, G. Cullia, S. Grasso and M. Zappalà, “The inhibition of cysteine proteases rhodesain and TbCatB: a valuable approach to treat human African trypanosomiasis,” Mini-Rev. Med. Chem., 2016; b. R. Ettari, A. Pinto, S. Previti, L. Tamborini, I. C. Angelo, V. La Pietra, L. Marinelli, E. Novellino, T. Schirmeister, M. Zappalà, S. Grasso, C. De Micheli and P. Conti, “Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation,” Bioorg. Med. Chem., vol. 23, pp. 7053-7060, 2015.
[4] A. Schmidt, H. Wu, R. E. MacKenzie, V. J. Chen, J. R. Bewly, J. E. Ray, J. E. Toth and M. Cygler, “Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase,” Biochemistry, vol. 39, pp. 6325-6335, 2000.
[5] T. C. Eadsforth, A. Pinto, R. Luciani, L. Tamborini, G. Cullia, C. De Micheli, L. Marinelli, S. Cosconati, E. Novellino, L. Lo Presti, A. Cordeiro da Silva, P. Conti, W. N. Hunter and M. P. Costi, “Characterization of 2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl ureido based inhibitors of TrypanosomabruceiFolD and testing for antiparasitic activity,” J. Med. Chem., vol. 58, no. 20, pp. 7938-7948, 2015.
[6] World Health Organization, “WHO guidelines for the treatment of Chlamydia trachomatis,” 2016. [Online]. Available: http://www.who.int/reproductivehealth/publications/rtis/chlamydia-treatment-guidelines/en/.
[7] a. K. Chambers, B. Judson and W. J. Brown, “A unique lysophospholipidacyltransferase (LPAT) antagonist, CI-976, affects secretory and endocytic membrane trafficking pathways,” J. Cell Sci., vol. 118, pp. 3061-3071, 2005; b. S. P. Ouellette and R. A. Carabeo, “A functional slow recycling pathway of transferrin is required for growth of Chlamydia,” Front. Microbiol.,vol. 1, 2010
Design, synthesis and biological characterization of PfGAPDH inhibitors
No full textMalaria has a tremendous health, social and economic impact on people living in tropical and subtropical regions of the world. WHO estimated 214 million cases and 438 000 deaths in 2015 and 3.2 billion people are globally at risk of infection1. Available treatments are becoming progressively less effective, mainly because parasites are constantly acquiring resistance toward the drug in use. Therefore, there is an urgent need of developing therapeutic agents acting on new targets.
Plasmodium parasites (P. falciparum is the most lethal), in their amastigote phase, produce energy only through glycolysis. In this pathway, GAPDH catalyses the first energy productive step and thus represent a valuable target for drug discovery. Known GAPDH inhibitors that bind to the active site are small nonselective molecules (iodoacetamide, 3-bromopyruvate), which irreversibly react with cysteine residues of the enzyme, including the catalytic one. We designed and synthesized the first series of inhibitors characterized by a 3-bromoisoxazoline warhead2, inspired by 3-bromoacivicin, a known inhibitor of reactive cysteine containing enzymes3. The compounds were assayed for the inhibitory activity on the P. falciparum isolated enzyme showing a biphasic irreversible inactivation. MS/MS studies of the digested protein demonstrated the mild reactivity of the exploited warhead, which only reacts with catalytic Cys (activated by a His residues). The human ortholog of GAPDH is only partially inhibited (up to 25%). We explained this through a negative cooperativity that prevents the alkylation of all the four monomers of hGAPDH, resulting in selectivity among the two isoforms of GAPDH4. Compounds were also assayed on cultures of the bloodstream form of P. falciparum and human cell lines in order to determine their in vitro antiparasitic activity and toxicity.
The goal of this project is to obtain compounds with improved potency and pharmacokinetic properties, suitable for in vivo studies on animal model of P. falciparum infections.
1. http://www.who.int/mediacentre/factsheets/fs094/en/
2. Bruno, S.; Pinto, A.; Paredi, G.; Tamborini, L.; De Micheli, C.; La Petra, V.; Marinelli, L.; Novellino, E.; Conti, P.; Mozzarelli, A. J. Med. Chem. 2014, 57(17), 7465-7471.
3. a) Conti, P.; Pinto, A.; Wong, P.E.; Major, L.L.; Tamborini, L.; Iannuzzi M.C.; De Micheli, C.; Barrett, M.P.; Smith, T.K. ChemMedChem 2011, 6(2), 329-333. b) Tamborini, L.; Pinto, A.; Smith, T.K.; Major, L.L.; Iannuzzi, M.C.; Cosconati, S.; Marinelli, L.; Novellino, E.; Lo Presti, L.; Wong, P.E.; Barrett, M.P.; De Micheli, C.; Conti, P. ChemMedChem 2012, 7(9), 1623-1634.
4. Bruno, S.; Margiotta, M.; Pinto, A.; Cullia, G.; Conti, P.; De Micheli, C.; Mozzarelli, A. Bioorg. Med. Chem. 2016, 24(12), 2654-2659
Folates in Trypanosoma brucei: achievements and opportunities
No full textTrypanosoma brucei is the agent of human African trypanosomiasis (HAT), a neglected disease that threatens the lives of 65 million people in sub-Saharan Africa every year. Unfortunately, available therapies are unsatisfactory, due primarily to safety issues and development of drug resistance. Over the last decades significant effort has been made in the discovery of new potential anti-HAT agents, with help from the World Health Organization (WHO) and private–public partnerships such as the Drugs for Neglected Diseases Initiative (DNDi). Whereas antifolates have been a valuable source of drugs against bacterial infections and malaria, compounds effective against T. brucei have not yet been identified. Considering the relatively simple folate metabolic pathway in T. brucei, along with results obtained in this research field so far, we believe that further investigations might lead to effective chemotherapeutic agents. Herein we present a selection of the more promising results obtained so far in this field, underlining the opportunities that could lead to successful therapeutic approaches in the future
Inspired by Nature : The 3-Halo-4,5-dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors
No full textOver the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re-emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3-chloro-4,5-dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3-halo-4,5-dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3-halo-4,5-dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors
Efficient synthesis of kainic acid analogues
No full textThe present paper deals with an improved synthesis of two molecular hybrids of AMPA and KA, compounds CIP-A and CIP-B, and their transformation into CIOP-A and CIOP-B, the corresponding amido derivatives. Exploiting the continuous-flow technology, a significant improvement in the synthesis of the glutamate agonists CIP-A and CIP-B was accomplished, in terms of overall yield, time, and excess of ethyl chlorooximinoacetate. Moreover, we find out the HPLC conditions suitable to separate, at a preparative level, the three intermediates formed in the 1,3-dipolar cycloaddition step
Synthesis and biological evaluation of new ligands designed to selectively interact with kainate receptors
No full textKainate receptors (KARs) are a family of ionotropic glutamate receptors (iGluRs). KARs are know to play a role in pain, epilepsy, neurodegenerative and psychiatric disorders but the lack of highly selective ligands for KAR subtypes (GluK1-5) prevented so far an exhaustive pharmacological characterization.1
Starting for the natural compound L-tricholomic acid, which is a non-selective AMPA/KA agonist, we designed a series of higher homologues of general structure A, in which the distal acidic group is linked to the isoxazoline ring through an aromatic spacer. Homologation is aimed at switching the profile from agonist to antagonist, by preventing the ligand binding domain closure, whereas an increased selectivity may arise from additional interactions played by the aromatic ring.
Similarly, starting from CIP-AS, a non selective AMPA/KA agonist previously developed by our group, we designed a series of higher homologues of general structure B. Moreover, we designed new analogs of general structure C, in which the isoxazoline ring was replaced by a N-substituted- pyrazoline, in order to explore the role played by an additional substituent (e.g. a methyl group) in this position, considering that, due to the presence of less hindered amino acids, KARs are known to accommodate larger substituents than AMPARs.
Preliminar biological data will be presented and discussed
Synthesis and pharmacological evaluation of conformationally constrained glutamic acid higher homologues
No full textHomologation of glutamic acid chain together with conformational constraint is a commonly used strategy to achieve selectivity towards different types of glutamate receptors. In the present work, starting from two potent and selective unnatural amino acids previously developed by us, we investigated the effects on the activity/selectivity profile produced by a further increase in the distance between the amino acidic moiety and the distal carboxylate group. Interestingly, the insertion of an aromatic ring as a spacer produced a low micromolar affinity NMDA ligand that might represent a lead for the development of a new class of NMDA antagonists
The inhibition of cysteine proteases rhodesain and TbCatB : A valuable approach to treat human african trypanosomiasis
No full textHuman African Trypanosomiasis (HAT) is an endemic parasitic disease of sub-Saharan Africa, caused by two subspecies of protozoa belonging to Trypanosoma genus: T. brucei gambiense and T. brucei rhodesiense. In this context the inhibition of the papain-family cysteine proteases rhodesain and TbCatB has to be considered a promising strategy for HAT treatment. Rhodesain, the major cathepsin L-like cysteine protease of T. brucei rhodesiense, is a lysosomal protease essential for parasite survival. It is involved in parasite invasivity, allowing it to cross the blood-brain barrier (BBB) of the human host, causing the second lethal stage of the disease. Moreover, it plays an important role in immunoevasion, being involved in the turnover of variant surface glycoproteins of the T. brucei coat and in the degradation of immunoglobulins, avoiding a specific immune response by the host cells. On the other hand TbCatB, a cathepsin B-like cysteine protease, present in minor abundance in T. brucei, showed a key role in the degradation of host transferrin, which is necessary for iron acquisition by the parasite. In this review article we now discuss the most active peptide, peptidomimetic and non-peptide rhodesain and TbCatB inhibitors as valuable strategy to treat HAT, due also to the complementary role of the two T. brucei proteases
Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenases
No full textCompounds based on the 3-Br-isoxazoline scaffold fully inhibit glyceraldehyde 3-phosphate dehydrogenase from Plasmodium falciparum by selectively alkylating all four catalytic cysteines of the tetramer. Here, we show that, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human ortholog was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer. The partial alkylation seems to produce a slow conformational rearrangement that severely limits the accessibility of the remaining active sites to bulky 3-Br-isoxazoline derivatives, but not to the substrate or smaller alkylating agents
