CA2686584A1 - Plasmodium proteases inhibitors and in silico screening methods for identifying the same - Google Patents

Abstract

The invention relates to the field of parasitology. Methods for screening method for identifying inhibitors of Plasmodium and compounds identified by these methods, including more particularly inhibitors of Plasmodium subtilisin-like proteases, are described. The invention also concerns anti-malaria compounds, anti-malaria compositions, and uses thereof for preventing, treating, improving, and/or alleviating a Plasmodium infection in a subject, and more Plasmodium vivax and/or by Plasmodium falciparum human infections.

Description

Our ref. : 273859.7 PLASMODIUM PROTEASES INHIBITORS AND
IN SILICO SCREENING METHODS FOR IDENTIFYING THE SAME
FIELD OF THE INVENTION

[0001] The invention relates to the field of parasitology. More particularly, it relates to the identification of inhibitors of Plasmodium, and to screening methods for identifying such inhibitors.

BACKGROUND OF THE INVENTION

[0002] Malaria is the most important human parasitic disease. More than forty percent of the world's population live in areas where malaria is transmitted (e.g., parts of Africa, Asia, the Middle East, Central and South America, Hispaniola, and Oceania). An estimated 700,000-2.7 million persons die of malaria each year, 75% of them being African children.

[0003] Biochemical and genetic analyses have shown that proteases of Plasmodium, the causative agent of malaria, play a central role in the entrance of the sporozoite and the merozoite into the host hepatocyte or red blood cell (RBC), respectively. The surface proteins of both extracellular invasive forms undergo obligatory proteolytic processing executed by parasite-encoded serine proteases, which are thus directly accessible to host factors such as antibodies or drugs. Importantly, 60% of the plasmatic proteins are protease inhibitors (mainly involved in the regulation of coagulation or complement activation) suggesting that the parasitic proteases active on the outer surface of the parasite are highly specific, differ from the host proteases and are insentive to host plasmatic protease inhibitors. Altogether, the features of the parasite serine proteases involved in RBC and hepatocytes invasion make them attractive targets as novel anti-malarials.

[0004] SUB2 and SUB1 are two essential Plasmodium serine proteases which are known to be involved in host cells invasion. The SUB2 subtilisin-like serine protease is discharged by the parasite onto the surface of the extracellular merozoite, where it performs proteolytic processing of major parasite surface proteins, a final maturation step that is essential for host cell invasion. SUB2 sequence is highly conserved in P.
falciparum and P.
vivax. Because of all its interesting properties, SUB2 has been described as a novel anti-Our ref. : 273859.7 malarial drug target in International PCT patent application W02006/120579.
The SUB1 enzyme has been shown to be involved in the egress of Plasmodium from infected erythrocytes and plays also a yet undefined role during the RBC invasion process per se.
SUB2 and SUB1 share substantial inter-species structural homology in their catalytic domains (e.g. >75% sequence identity between the PfSUB2 and PvSUB2 domains, and between PfSUB1 and PvSUB1 domains). The Plasmodium genome harbours a third prokaryotic subtilisin-like serine protease, SUB3, which differs from SUB1 and SUB2 in being not essential for the intra-erythrocytic cycle. However, its expression is activated after the entry of the sporozoites into the hepatocytes, suggesting a role during the establishment of the infectious process in mammalian hosts.

[0005] Chloroquine is a 4-aminoquinoline drug used in the treatment or prevention of malaria. Popular drugs based on chloroquine phosphate (also called nivaquine) are Chloroquine FNA, Resochin and Dawaquin. Worryingly, resistance to both Plasmodium falciparum and P. vivax, the two main species infecting humans, have eroded treatment efficacy and malaria control measures. In addition, mosquito resistance to insecticides is spreading. Efforts at developing a malaria vaccine with long term efficiency has met with limited success.

[0006] There is thus an urgent need for the discovery and development of novel anti-malarials. There is also a need for compounds targeting Plasmodium invasion process of either the hepatocyte or the red blood cells. There is also a need for enzyme inhibitors effective for prophylaxis preventing host infection.

BRIEF SUMMARY OF THE INVENTION

[0007] The present inventors have designed methods for screening inhibitors of Plasmodium, and more particularly inhibitors of Plasmodium subtilisin-like proteases. The inventors have also identified new inhibitors of Plasmodium, and more particularly inhibitors of Plasmodium subtilisin-like proteases.

Our ref. : 273859.7 [0008] One particular aspect of the invention relates to a screening method for identifying inhibitors of Plasmodium and compounds identified using such methods, including more particularly inhibitors of Plasmodium subtilisin-like proteases.

[0009] Another aspect of the invention concerns anti-malaria compounds, and more particularly compounds inhibiting a Plasmodium protease. These compounds may be advantageously identified by the screening method of the invention. Preferably the Plasmodium protease is a subtilisin-like protease. In various embodiments the subtilisin-like protease is SUB1, SUB2 or SUB3.

[00010] A related aspect concerns pharmaceutical composition comprising a compound as defined herein. In preferred embodiments, the pharmaceutical composition is formulated as an anti-malarial drug (e.g. prophylaxis and/or treatment of Plasmodium vivax and/or by Plasmodium falciparum infections).

[00011] The invention is also directed to methods for preventing, treating, improving, and/or alleviating a Plasmodium infection in a subject. The method comprises administering to the subject a therapeutically effective amount of a compound or of a pharmaceutical composition as defined herein.

[00012] The invention also relates to nucleic and amino acid sequences as shown in Figures 1C, 5A, 5B, 6A, 6B, 7A and 7B. In particular aspects the invention relates to the Plasmodium vivax Belem strain SUB1 wild-type and its amino acid sequence as defined at Figure 1C and encoded by the polynucleotide of sequence as set forth in Figure 5A. The invention further relates to recombinant PvSUB1, PfSUB1 and PbSUB1 purified enzymes and to the recodoned nucleic sequences of PfSUB1 and PbSUB1 as defined at Figure 6A
and 7A, respectively.

[00013] The invention also relates to FRET substrates as described herein and as shown in Figure 2B, and the uses thereof in high throughput screening methods and screening assays. Related aspect concerns throughput screening methods, including fluorescence based methods for identifying inhibitors of Plasmodium, including inhibitors of Plasmodium subtilisin-like proteases.

Our ref.: 273859.7 [00014] Additional aspects, advantages and features of the present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are exemplary and should not be interpreted as limiting the scope of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
1. SCREENING METHODS

[00015] One aspect of the invention concerns screening methods for identifying inhibitors of Plasmodium, and more particularly inhibitors of Plasmodium subtilisin-like proteases.

[00016] According to a particular aspect, the invention relates to a screening method which comprises:
(a) in silico docking a 3D structure of a plurality of test compounds to a 3D
model of one or more Plasmodium protease (e.g. a subtilisin-like protease); and next carrying out at least one of the steps of:
(b) testing in vitro and/or ex vivo test compound(s) from step (a) having a desired in silico docking activity;
(c) testing in vivo test compound(s) from step (a) having a desired in silico docking activity;
(d) testing in vivo test compound(s) from step (b) having a desired in vitro and/or ex vivo activity.

[00017] According to another aspect, the invention relates to a screening method for identifying inhibitors of multiple Plasmodium species. In one embodiment the method comprises:
(a) in silico docking a 3D structure of a plurality of test compounds to a 3D
model of one or more Plasmodium protease (e.g. subtilisin-like proteases); and (b) testing in vitro, ex vivo and/or in vivo test compounds from step (a) having a desired in silico docking activity;

Our ref. : 273859.7 wherein the step (b) is carried out for identifying inhibitors active against one or more Plasmodium species which is(are) different from the one or more Plasmodium protease of step (a).

[00018] A further aspect, the invention relates to a method for identifying inhibitors of Plasmodium falciparum, comprising: (i) a first screening step directed in identifying potential inhibitors of Plasmodium vivax, and (ii) testing subsequently in vitro, ex vivo and/or in vivo potential inhibitors from step (i) for inhibition against Plasmodium vivax, Plasmodium falciparum and/or Plasmodium berghei. The first screening step comprises a step that is carried out in silico, more preferably by in silico docking a 3D structure of potential inhibitors into a 3D model of a Plasmodium vivax protease. In preferred embodiments the Plasmodium vivax protease is a recombinant SUB1 protease, for instance a recombinant SUB1 protease comprising the amino acid sequence of PvSUB1-Bellem as defined at Figure 1C.

[00019] The screening methods of the invention comprise a step that is carried out in silico. In silico screening of drugs and in silico-based drug design is becoming more and more popular (e.g. Song et al., 2005, PNAS, 102:4700-05; Plewczynski et al., 2007, Chem Biol Drug Des, 69:269-279; Leitao et al., 2008, Eur J Med Chem, 43:1412-1422;
Kirchmair et al, 2008, Curr Med Chem 15:2040-53; Zoete et al., 2009, J Cell Mol Med, 13:238-78; Jain AN 2004, Curr Opin Drug Discov Devel, 7:396-403; Rester U 2008, Curr Opin Drug Discov.
Devel 11:559-68). The present invention uses general principles of in silico screening known and applied by those skilled in the art in the discovery or screening of enzymes inhibitors, including protease inhibitors. Without being bound by any particular details or explanation, a first element which is typically required is a virtual 3D-structure of the targeted protein. Such structure may be obtained from the 3D X-ray crystallography resolution, or from a model deriving from the 3D X-ray crystallography resolved structure of one or more closely related proteins. The second required element is a precise spatial identification of the catalytic site of interest (e.g. hydrophobic pocket). Such precise spatial identification generally comprises 3D coordinates of (i) the catalytic site where the substrate will dock and (ii) of the proximal amino acids which participates in the docking because of physico-chemical forces (e.g.
hydrophobic interactions, hydrogen bonding, van der Waals forces, etc.).
Finally, the third required element is the 3D structure of the chemical compounds to be tested (e.g. a library Our ref. : 273859.7 of chemical compounds). 3D structure of a chemical compound may be a X-ray crystallography resolved structure or a 3D structure which has been modeled using the 2D
chemical structure or the chemical formula of the compound. Having these three elements in silico screening typically takes place by using computational chemistry software, the software calculating, for each chemical compound to be tested, probabilities for the compound to interact or bond into the catalytic site of the targeted protein.
Compounds with the best score are selected for subsequent in vitro, ex vivo, and/or in vivo rounds of screening. Suitable computational chemistry software include, but are not limited to, FIexTMFlexX-PharmTM, and IcmTM

[00020] The Plasmodium protease may be selected from SUB1, SUB2 and SUB3. One may take advantage of the similarity of Plasmodium subtilisins active site and use, as the 3D
model, a homology model of two different Plasmodium proteases (e.g. SUB1 and SUB2).
Similarly, one may take advantage of the similarity of SUB1 active site with bacterial subtilisins and use, as the 3D model, a homology model based on known bacterial and/or fungi subtilisins 3D structures. Examples of known and accessible 3D
structures include, but are not limited to, those published in the RCSB Protein Data BankTM that are directly accessible on the web site pdb.org or via the NCBI web site. Particular examples include the following proteins: BPN' (Acc. No. 1 LW6); sphericase (Acc. No. 1 EA7);
Thermitase (Acc.
No. 2TEC); AK-1 Serine protease (Acc. No. 1 DBI); subtilisin Carlsberg (Acc.
No. 1 ROR);
proteinase K (Acc. No. 1 IC6) and Bacillus lentus subtilisin (Acc. No. 1 GCI).

[00021] According to some embodiments, the in silico, in vitro, ex vivo and in vivo testing may be carried out on different strains of Plasmodium and/or by using different Plasmodium proteases. For instance, according to one embodiment the in silico docking step involves a protease from Plasmodium vivax whereas in vitro, ex vivo and/or in vivo testing involves a protease from Plasmodium vivax, Plasmodium falciparum and/or Plasmodium berghei, respectively.

[00022] The invention encompasses methods for identifying anti-malarial candidates targeting a set of Plasmodium enzymes crucial for the parasite invasion into and egress from host cells processes.

Our ref.: 273859.7 [00023] The invention encompasses methods for identifying compounds capable of targeting more than one enzyme, presumably at different parasite stages, which is likely to maximize efficacy, and minimize the risks of failure or resistance.

[00024] In embodiments, the selected Plasmodium proteases belong to the same family of enzymes, thus displaying common features in their active sites, thereby providing the possibility of identifying biologically active inhibitors capable of binding multiple Plasmodium targets.

[00025] The invention further encompasses assay kits and methods for screening of possible therapeutic anti-malarial compounds and compositions to help alleviate, treat and/or prevent Plasmodium infections, especially in humans.

II. THERAPEUTICS

[00026] As exemplified hereinafter the methods of the invention successfully resulted in the identification of compounds having anti-malarial activity, in vitro, in vitro, ex vivo and in vivo. In the context of the present invention, anti-malaria compounds, Plasmodium-inhibiting compounds, inhibitors of Plasmodium and anti-malarial candidates are equivalent terms (have the same meaning).

[00027] According to one aspect, the compounds of the invention are defined by the Formula I or Formula II:

R~ H R1 n n I II
or a pharmaceutically acceptable salt thereof, wherein Our ref. : 273859.7 nis0or1;
A is a heteroaryl ring fused to the phenyl ring, the heteroaryl ring being optionally substituted with one or more R3 substituents;

R1 is 1) aryl, 2) heteroaryl, or 3) heterocyclyl;
R2 is 1) C(O)CH2-heteroaryl substituted with C(O)NH-aryl, or 2) heterocyclyl substituted with one or more aryl, the aryl being substituted with one or more R4 substituents;
R3 is 1) Cl-C6 alkyl optionally substituted with one or more C(O)heterocyclyl or aryl, the aryl being optionally substituted with one or more halo groups, 2) aryl substituted with C(O)NH-aryl, or 3) NH-aryl substituted with 0C1-C6 alkyl; and R4 is 1) halogen, or 2) OC1-C6 alkyl.

[00028] As used herein, the term "alkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, for example, C1-C6 as in C1-C6 alkyl is defined as including groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement. Examples of C,-C6-alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl and hexyl.

[00029] As used herein, the term "halo" or "halogen" is intended to mean fluorine, chlorine, bromine and iodine.

[00030] As used herein, the term "aryl", either alone or in combination with another radical, means a carbocyclic aromatic monocyclic group containing 6 carbon atoms which may be further fused to a second 5- or 6-membered carbocyclic group which may be Our ref. : 273859.7 aromatic, saturated or unsaturated. Aryl includes, but is not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl and tetrahydronaphthyl.

[00031] As used herein, the term "heteroaryl" is intended to mean a monocyclic or bicyclic ring system of up to ten atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of 0, N, and S.
The heteroaryl substituent may be attached either via a ring carbon atom or one of the heteroatoms.
Examples of heteroaryl groups include, but are not limited to thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, and thiazolo[4,5-b]-pyridine.

[00032] As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" is intended to mean a 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of 0, N and S. Examples of heterocycles include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl.

[00033] As used herein, the term "heterobicycle" either alone or in combination with another radical, is intended to mean a heterocycle as defined above fused to another cycle, be it a heterocycle, an aryl or any other cycle defined herein. Examples of such heterobicycles include, but are not limited to, coumarin, benzo[d][1,3]dioxole, 2,3-dihydrobenzo[b][1,4]dioxine and 3,4-dihydro-2H-benzo[b][1,4]dioxepine.

[00034] As used herein, the term "optionally substituted with one or more substituents" or its equivalent term "optionally substituted with at least one substituent" is intended to mean that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. The definition is intended to mean from zero to five substituents.

Our ref. : 273859.7 [00035] The invention encompasses pharmaceutically acceptable salt including acid addition salts, and base addition salts. As used herein, the term "pharmaceutically acceptable salt" is intended to mean those salts which retain the biological effectiveness and properties of the free acids or bases, which are not biologically or otherwise undesirable.
Desirable are salts of a compound are those salts that retain or improve the biological effectiveness and properties of the free acids and bases of the parent compound as defined herein or that takes advantage of an intrinsically basic, acidic or charged functionality on the molecule and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are also described, for instance, in Berge et al., "Pharmaceutical Salts", J.
Pharm. Sci. 66, 1-19 (1977).

[00036] The compounds of the present invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined in terms of absolute stereochemistry, such as (R)-- or (S)--or, as (D)- or (L)-for amino acids. The present invention is intended to include all such possible isomers, as well as, their racemic and optically pure forms. Certain compounds of the present invention may exist in Zwitterionic form and the present invention includes Zwitterionic forms of these compounds and mixtures thereof.

[00037] In general, all compounds of the present invention may be prepared by any conventional methods, using readily available and/or conventionally prepayable starting materials, reagents and conventional synthesis procedures.

[00038] According to some embodiments, the compound is selected from the following table:
Compound # Structure MW (Daltons) C/D3 H / \ 422 NH
H

Our ref. 273859.7 H

N :lap A/G 1 q 432 O NH
O /

N
N NH

N ~N
O

CI

Our ref. : 273859.7 [00039] According to some embodiments, the compound is selected from the following list:

o N

O
C'I

o O l o O
O
N N
N
N

Our ref. 273859.7 N

O

CE
N
N O

rl N
C~~Qj HO O

Our ref. 273859.7 H F F - I \
H N \ I O, \ / /
N N - `N
H O \ /N 1 ` HO I H
i t H I' N\ H
\ N i \ O, O
O

H I\
\ /
N O. \ / `N
N H \ .~ \ ~H
H
Br / I H
O N NiN\ I O. F O
HO
H - - I , O N i AN' H\ o' N ~ H H
O
I IJH I p F
~ \ ~ O. I ~ I \
N N I
O H N
N H I~\ H O N
\ I i N I O, CI'~J I H
N N

N. I b \
N N CI - /
H \ / `N
N H `N O`` ~I\ N\ H
\ NI\ h__ t \ H r 0 N \ CI HO
Br H

1iI 0 \ HO qN
N
\N
\ / O N CI \ `N NH

H H ' H O DN / O' N H \/ Icl cl/
N" N" CI J-H 0"
H ov J
i i \ I J O-N

O

N H CI HO \
/
NI N 0 \ / / H
O N H\ CI \ H 0 I c H
Br CI N 0. 0 OH N N\ F
H

0 l; H

Our ref. : 273859.7 [00040] In a related aspect, the invention concerns to pharmaceutical composition comprising a compound as defined herein, and more particularly compositions formulated as an anti-malarial drugs. The invention further relates to the use of a compound as defined herein for the manufacture of a medicament for preventing and/or treating malaria.

[00041] The invention also encompasses the uses of a compound of the invention as defined herein, in combination with one or more existing anti-malarial drug (see hereinafter).

[00042] According to some embodiments, the compounds and compositions of the invention can prevent, reduce and/or inhibit the Plasmodium parasite infection process, i.e.
reduces or inhibits the Plasmodium parasite egress from and/or invasion into host cells. In preferred embodiment, the parasite is Plasmodium vivax or Plasmodium falciparum.

[00043] According to some embodiments, the compounds and compositions of the invention are capable of targeting more than one enzyme, presumably at different parasite stages, thereby maximizing efficacy, and/or minimizing risks of failure or resistance.
Preferably, the compounds inhibit the activity of at least one subtilisin-like protease, more preferably, SUB1, SUB2 and/or SUB3.

[00044] According to some embodiments, the compounds and compositions of the invention are capable of inhibiting Plasmodium resistant strains, including but not limited to strains resistant to chloroquin, strains resistant to artemisinin, and/or strains resistant to derivatives of such anti-malarial drugs.

[00045] In preferred embodiments the compounds of the invention have Ki less than about 50 pM on recombinant subtilisin-like protease, and in more preferred embodiments less than 10 pM. In other preferred embodiments the compounds of the invention have an IC50 of about 20 pM or less, of about 1 pM or less, or about 100 nM or less.
In some embodiments the compounds of the invention have an in vivo LD50 (in humans or animals) of about 33 mg/kg or less (e.g. <_ 30 mg/kg, <_ 10 mg/kg, or <_ 1 mg/kg).

Our ref. : 273859.7 [00046] In a related aspect, the invention concerns to a method for preventing, treating, improving, and/or alleviating a Plasmodium infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound or of a pharmaceutical composition as defined herein.

[00047] The term "subject" includes living organisms in which a Plasmodium infection can occur. The term "subject" includes animals (e.g., mammals, e.g., cats, dogs, horses, pigs, cows, goats, sheep, rodents, e.g., mice or rats, rabbits, squirrels, bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans)), as well as wild and domestic bird species (e.g. chickens), and transgenic species thereof. Preferably, the subject is a mammal. More preferably, the subject is a human.

[00048] The pharmaceutical compositions of the invention may comprise a therapeutic agent (e.g. a compound of Formula I or II as defined herein or a compound identified by the above screening method) in a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes.

[00049] The pharmaceutical compositions of the invention may comprise a compound of the invention as defined herein, in combination with one or more existing anti-malarial drug, including but not limited to chloroquine FNA, resochin, dawaquin, artemisinin, quinine, amodiaquine, sulfadoxynie, pyrimethamine, mefloquine, proguanil, artesunate, halofantrine, and atovaquone.

[00050] With respect to pharmaceutically useful compounds or compositions according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount"
(as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefits to the individual.

[00051] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, Our ref. : 273859.7 claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLE

[00052] EXAMPLE 1: An in silico screening approach to select inhibitors of Plasmodium [00053] Red blood cell egress and invasion by Plasmodium parasites strictly depend upon the precise maturations of parasite proteins SERA5, a cystein protease implicated in the rupture of the parasitophorous vacuole membrane and MSP1 (Merozoite Surface Protein 1). The parasite subtilisin-like serine protease SUB1 plays a key role in the process (S. Yeoh et al, Cell, 131(6), 1072-83 (2007)) as it is essential for the merozoite egress. On the other hand, SUB2, another subtilisin-like serine protease is essential for the meroxoite entry into RBC. Taking advantage of the similarity of SUB1 active site with bacterial subtilisins, we have used an in silico screening approach and have identified inhibitors of Plasmodium.

[00054] The general strategy for screening and validation of Plasmodium inhibitors is summarized in Figure IA. Briefly, commonly used filters were applied to a commercial chemical library comprising more than 600 000 compounds to identify compounds having a drug like structures (e.g. Lipinski's rules). Remaining compounds (149 992) were then screened by using an in silico approach. As described hereinafter, in silico hits (366) were purchased and tested in the laboratory for Ki determination (11 compounds), determination (5 compounds) and in vivo evaluation (1 compound).

[00055] The in silico step was based on in silico docking of test compounds into SUB1 modeled active sites, and more particularly a 3D model of a recombinant SUB1 protein (PvSUB1) derived from Plasmodium vivax sequences.

[00056] The selection and optimization of the PvSUB1 optimized model is illustrated in Figure 1 B. That figure (top) shows that the final 3D structure was obtained from conserved amino acid sequences surrounding D15, H58, N153, S221 which are involved in the Our ref. : 273859.7 calalytic cleavage site of PvSUB1. The bottom Figure 1 B shows a representative example of the iterative computerized process for obtaining a 3D model of the catalytic site of PvSUB1.
The 3D structures of test compounds was also inputted and tested for docking into the active site by using different computer software (e.g. FIexTM, FlexX-PharmTM, IcmTM). Those with the best score were selected for the subsequent screening step.

[00057] It is the amino acid sequence of PvSUB1 of Plasmodium vivax Bellem isolate which was used for creating the PvSUB1 optimized model described hereinabove.
Figure IC shows multiple alignment of Plasmodium berghei-SUB1, Plasmodium yoelii-SUB1, Plasmodium vivax (clone Sall)-SUB1, Plasmodium falciparum (clone 3D7)-SUB1 (all obtained from www.plasmodb.org) and Plasmodium vivax (clone Belem)-SUB1 protein sequences. Grey and black boxes stand for similar and identical conserved residues respectively. Predicted signal peptide sequences are boxed. Predicted (PySUB1, PvSUB1-Sall) and experimentally identified (PfSUB1-3D7, PbSUB1 and PvSUB1-Belem) auto-maturation activation sites are indicated by a vertical arrow. The four essential residues forming the catalytic site, Aspartate (D), Histidine (H), Asparagine (N) and Serine (S) are in red boxes. The Table displays percentages of similarity and identity between the full length and enzymatic forms of all the SUB1 orthologues.

[00058] The PvSUB1, PfSUB1 and PbSUB1 recombinant purified enzymes expressed using the baculovirus/insect cells expression system, in combination with a FRET assay, were used for Ki determination. The nucleotide and the amino acid sequences of each of PvSUB1, PfSUB1 and PbSUB1 are shown in Figures 5A to 7B. The nucleotide sequences of PISUBI and PbSUB1 was "recodoned" for avoiding codon bias of the Plasmodium open reading frames compared to other organisms, including insect cells.

[00059] Briefly, SUB1 proteins exist under a pro-form (80 kDa) and an active form (48-50 kDa). Figure 2A shows that the HPLC-fractions containing the recombinant baculovirus-expressed PvSUB1 (48-50 Da bands on the CoomassieTM colored gel) display a significant activity on SUB1-specific FRET fluorescent substrates, thereby demonstrating that the recombinant purified PvSUB1 is an active enzyme.

Our ref. : 273859.7 [00060] Figure 2B depicts the FRET-based specific enzymatic assay which was used. In this particular case the assay consists in measuring fluorescence of tagged peptidic substrate of SUB1. An uncleaved peptide has low intensity emission whereas, when the peptide is cleaved by SUB1, high intensity emissions are measured. Bottom table of Figure 2C shows the different substrates that were used (amino acid sequence, the quencher and the fluorofore (Dabcyl/EDANS or Far Red)). Eleven molecules where thus tested and their Ki (inhibition constant) was determined on the PvSUB1 recombinant enzyme. Figure 2C provides exemplary curves for the compound C/D3 (DMSO was the negative control). Out of the eleven molecules, five with a Ki less than 50 pM
were retained for determination of their anti-parasite effect (IC50) on the in vitro culture of the chloroquino-sensitive (3D7) and chloroquino-resistant (Dd2) P. falciparum clones according to Desjardins et al. (Desjardins et al. Antimicrob Agents Chemother. 1979 Dec;16(6):710-8).
The results are presented in Table 1 hereinafter. The fact that there is a very good correlation between Ki and IC50 for the compounds tested suggests that the anti-parasitic effect observed is likely to be the consequence of the inhibition of the PfSUB1 enzyme.C/D3 was the most active compounds.

[00061] Next, the five remaining SUB1 inhibitors were evaluated biologically for their impact on P. falciparum 3D7 stage-specific ex vivo culture. Figures 3A and 3B
show general principles and results of an invasion test in presence of the compounds at 90 pM
(samples were taken at T=12 h). The results show the inhibition of the SERA 5 maturations and of the egress of the merozoites using YOYO1 Facs technique (Li et al, Cytometry, 71A, 297-307, 2007). As seen in western blots at bottom of Figure 3A, the maturation of SERA5 by SUB1 successive cleavages is inhibited by the five test compounds, while the presence of DMSO or of an inhibitor of another protease (E64) are inactive on SERA5 maturation. As seen in Figure 3B, the two tested compounds (A/G6 and C/D3) were able to maintain the parasites to the schizonts stage while, with the negative control (DMSO), the parasites evolved to the subsequent ring stage. Since PISUB1 is known to be crucial for the merozoite egress, and since PvSUB1 inhibitors affect P. falciparum merozoites egress, these results demonstrate that PvSUB1 inhibitors also inhibit endogenous PfSUB1 enzyme ex vivo, thus explaining the anti-P. falciparum activity of the compounds.

Our ref. : 273859.7 [00062] Finally, C/D3 was tested in vivo on P. berghei-infected mice. As shown in Figure 4, the compound inhibited red blood cell infection in a dose-dependent manner. It was estimated that C/D3 has a LD50 of about 33 mg/kg (LD50 of chloroquine is about 2 mg/kg). C/D3 showed no obvious signs of toxicity.

[00063] Altogether these results demonstrate that targeting a Plasmodium vivax therapeutic target leads to the selection and the validation of chemical compounds having a potent activity against different Plasmodium species, which are responsible for the severe forms of malaria. Therefore the screening methods and the chemical compounds described herein are potentially useful in anti-malaria therapy and prophylaxis against at least the two main Plasmodium infecting humans: P.vivax and P.falciparum.

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Our ref. : 273859.7 [00064] Headings are included herein for reference and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[00065] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.
Thus, for example, reference to "a compound" includes one or more of such compounds, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[00066] Unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible.
Any numerical value, however, inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such.

[00067] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims (26)

1. A compound of Formula I or Formula II:

or a pharmaceutically acceptable salt thereof, wherein n is 0 or 1;
A is a heteroaryl ring fused to the phenyl ring, the heteroaryl ring being optionally substituted with one or more R3 substituents;

R1 is 1) aryl,

2) heteroaryl, or

3) heterocyclyl;
R2 is 1) C(O)CH2-heteroaryl substituted with C(O)NH-aryl, or 2) heterocyclyl substituted with one or more aryl, the aryl being substituted with one or more R4 substituents;

R3 is 1) C1-C6 alkyl optionally substituted with one or more C(O)heterocyclyl or aryl, the aryl being optionally substituted with one or more halo groups, 2) aryl substituted with C(O)NH-aryl, or 3) NH-aryl substituted with OC1-C6 alkyl; and R4 is 1) halogen, or 2) OC1-C6 alkyl.

2. The compound of claim 1, wherein said compound is selected from the group consisting of:

3. The compound of claim 1, wherein said compound inhibits a Plasmodium protease.

4. The compound of claim 3, wherein said Plasmodium protease is a subtilisin-like protease.

5. The compound of claim 4, wherein the subtilisin-like protease is SUB1, SUB2 or SUB3.

6. The compound of claim 1, wherein said compound prevents, reduces or inhibits the Plasmodium parasite egress from and/or invasion into host cells.

7. The compound of claim 1, wherein said compound inhibits at least two Plasmodium proteases at different stages of the Plasmodium parasite life cycle process.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7.

9. The pharmaceutical composition according to claim 8, wherein said composition is formulated as an anti-malarial drug.

10. Use of compound according to any one of claims 1 to 7 or of a pharmaceutical composition according to claim 8 or 9, for the prevention or treatment of malarial infection.

11. The use of claim 10, wherein the malarial infection is an infection by Plasmodium vivax or by Plasmodium falciparum.

12. A method for preventing, treating, improving, and/or alleviating a Plasmodium infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 7 or of a pharmaceutical composition according to claim 8 or 9,.

13. The method of claim 12, wherein the subject is a human and wherein the Plasmodium infection is an infection by Plasmodium vivax or by Plasmodium falciparum.

14. A screening method for identifying inhibitors of Plasmodium, comprising:
(a) in silico docking a 3D structure of a plurality of test compounds to a 3D
model of one or more Plasmodium protease; and next carrying out at least one of the steps of:
(b) testing in vitro and/or ex vivo test compound(s) from step (a) having a desired in silico docking activity;
(c) testing in vivo test compound(s) from step (a) having a desired in silico docking activity;
(d) testing in vivo test compound(s) from step (b) having a desired in vitro and/or ex vivo activity.

15. The method of claim 14, wherein the Plasmodium protease at step (a) is different from the Plasmodium protease at step (b) and/or (c).

16. The method of claim 15, wherein the Plasmodium protease at step (a) is from Plasmodium vivax whereas the Plasmodium protease at step (b) is from Plasmodium vivax or Plasmodium falciparum and wherein the Plasmodium protease at step (c) or (d) is from Plasmodium berghei

17. The method of claim 15, wherein the 3D model of one or more Plasmodium protease is a single homology model of at least two Plasmodium proteases.

18. The method of claim 17, wherein said at least two Plasmodium proteases consists of SUB1 and SUB2.

19. The method of claim 14, wherein said in vitro testing comprises measuring for said test compound an inhibition constant (Ki) and/or an IC50.

20. The method of claim 14, wherein said in vivo testing comprises measuring for said test compound red blood cell infection in a Plasmodium berghei-infected mice.

21. A screening method for identifying inhibitors of multiple Plasmodium species, comprising:
(a) in silico docking a 3D structure of a plurality of test compounds to a 3D
model of one or more Plasmodium protease; and (b) testing in vitro, ex vivo, and/or in vivo test compound from step (a) having a desired in silico docking activity;
wherein said step (b) is carried out for identifying inhibitors active against one or more Plasmodium species which is(are) different from the one or more Plasmodium protease of step (a).

22. A method for identifying inhibitors of Plasmodium falciparum, comprising:
(i) a first screening step directed in identifying potential inhibitors of Plasmodium vivax, and (ii) testing subsequently in vitro, ex vivo and/or in vivo potential inhibitors from step (i) for inhibition against Plasmodium vivax, Plasmodium falciparum and/or Plasmodium berghei.

23. The method of claim 22, wherein said first screening step comprises a step that is carried out in silico.

24. The method of claim 22 or 23, wherein said screening step comprises in silico docking a 3D structure of said potential inhibitors into a 3D model of a Plasmodium vivax protease.

25. The method of claim 24, wherein the Plasmodium vivax protease is a recombinant SUB1 protease.

26. The method of claim 25, wherein the recombinant SUB1 protease comprises the amino acid sequence of PvSUB1-Bellem as defined at Figure 1C.