Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246-252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.     

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.     

Optimization of plasmepsin inhibitor by focusing on similar structural feature with chloroquine to avoid drug-resistant mechanism of Plasmodium falciparum

The plasmepsins are specific aspartic proteases of the malaria parasite and a potential target for developing new antimalarial agents. Our previously reported peptidomimetic plasmepsin inhibitor with modified 2-aminoethylamino substituent, KNI-10740, was tested against chloroquine sensitive Plasmodium falciparum, D6, to be highly potent, however, the inhibitor exhibited about 5 times less activity against multi-drug resistant parasite (TM91C235). We hypothesized the potency reduction resulted from structural similarity between 2-aminoethylamino substituent of KNI-10740 and chloroquine. Then, we modified the moiety and finally identified compound 15d (KNI-10823), that could avoid drug-resistant mechanism of TM91C235 strain.     

High-affinity inhibition of a family of Plasmodium falciparum proteases by a designed adaptive inhibitor

Drug development against viral or microbial targets is often compounded by the existence of naturally occurring polymorphisms or drug resistant mutations. In the case of Plasmodium falciparum, the etiological agent of malaria, four related and essential proteases, plasmepsin I, II, and IV and the histo-aspartyl protease (HAP), have been identified in the food vacuole of the parasite. Since all of these enzymes are involved in the hemoglobin degradation of infected victims, the simultaneous inhibition of the four enzymes can be expected to lead to a faster starvation of the parasite and to delay the onset of drug resistance, since four enzymes will need to mutate in a concerted fashion. This study describes the design of an adaptive inhibitor intended to inhibit the entire plasmepsin family. Adaptive inhibitors bind with extremely high affinity to a primary target within the family and maintain significant affinity against the remaining members. This objective is accomplished by engineering the strongest and most specific interactions of the inhibitor against conserved regions of the binding site and by accommodating target variations by means of flexible asymmetric functional groups. Using this approach, we have designed an inhibitor with subnanomolar affinity (0.5 nM) against the primary target, plasmepsin II, and with no loss or a very small loss of affinity against plasmepsin IV, I, and HAP (K(i) ratios of 0.4, 7.1, and 17.7, respectively). The core of the inhibitor is defined by an allophenylnorstatine scaffold. Adaptability is provided by an asymmetric amino indanol functional group facing one of the key variable regions in the binding site. Adaptive inhibitors, which display high affinity against several variations of a primary target, are expected to play an important role in the chemotherapy of infectious diseases.     

Solid-phase library synthesis of reversed-statine type inhibitors of the malarial aspartyl proteases plasmepsin I and II

With the aim to develop inhibitors of the plasmepsin I and II aspartic proteases of the malaria parasite Plasmodium falciparum, we have synthesized sets of libraries from novel reversed-statine isosteres, using a combination of solution phase and solid phase chemistry. The synthetic strategy furnishes the library compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The products were evaluated for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits an in vitro activity of 28% inhibition of plasmepsin II at an inhibitor concentration of 0.5 microM (K(i) for Plm II=5.4 microM).     

The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.     

New inhibitors of the malaria aspartyl proteases plasmepsin I and II

New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are described. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits K(i) values of 250 nM and 1.4 microM for Plm I and II, respectively.     

Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin

Plasmepsin (Plm) is a potential target for new antimalarial drugs, but most reported Plm inhibitors have relatively low antimalarial activities. We synthesized a series of dipeptide-type HIV protease inhibitors, which contain an allophenylnorstatine-dimethylthioproline scaffold to exhibit potent inhibitory activities against Plm II. Their activities against Plasmodium falciparum in the infected erythrocyte assay were largely different from those against the target enzyme. To improve the antimalarial activity of peptidomimetic Plm inhibitors, we attached substituents on a structure of the highly potent Plm inhibitor KNI-10006. Among the derivatives, we identified alkylamino compounds such as 44 (KNI-10283) and 47 (KNI-10538) with more than 15-fold enhanced antimalarial activity, to the sub-micromolar level, maintaining their potent Plm II inhibitory activity and low cytotoxicity. These results suggest that auxiliary substituents on a specific basic group contribute to deliver the inhibitors to the target Plm.     

Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum

A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.     

Molecular modeling and prediction of binding mode and relative binding affinity of Art-Qui-OH with P. falciparum Histo-Aspartic Protease (HAP)

The relative binding affinity in terms of ΔΔG _bind-cald value of the antimalarial compound artemisinin-quinine hybrid is primarily derived and is discussed in this article with reference to the ΔG _bind-cald values of two known inhibitors Pepstatin-A and KNI-10006 complexed with HAP enzyme. The ΔG _bind-cald value for KNI-10006 and Pepstatin-A is -14.10 kcal/mol and -13.09 kcal/mol respectively. The MM-GB/SA scoring results in the relative binding energy (ΔΔG _bind-cald) of the hybrid molecule with respect to Pepstatin-A as 2.43 kcal/mol and 3.44 kcal/mol against KNI-10006. The overall binding mode of Art-Qui-OH resembles that of Pepstatin-A binding in HAP active site. We suggest here that the ΔΔG _bind-cald value & proposed binding mode of the Art-Qui-OH for HAP enzyme should be considered for further structure-based drug design effort.     

A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV-1 protease

The efficacy of HIV-1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI-764, a second-generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI-577 differs from KNI-764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV-1 protease have been determined by X-ray crystallography at 2.0 A resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI-764 to adapt to the mutated binding site cavity more readily than KNI-577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug-resistant mutant protease; however KNI-764 gains binding entropy while KNI-577 loses binding entropy. The gain in binding entropy by KNI-764 accounts for its low susceptibility to the drug-resistant mutation. The heat capacity change associated with binding becomes more negative when KNI-764 binds to the mutant protease, consistent with increased desolvation. With KNI-577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI-764 when it is bound to the drug-resistant mutant. The opposite is observed for KNI-577. Consistent with these observations, it appears that KNI-764 is able to gain binding entropy by a two-fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease.     

Inhibitor binding to the plasmepsin IV aspartic protease from Plasmodium falciparum

Plasmepsin IV (Plm IV) is one of the aspartic proteases present in the food vacuole of the malaria parasite Plasmodium falciparum involved in host hemoglobin degradation by the parasite. Using a series of previously synthesized plasmepsin inhibitors [Ersmark, K., et al. (2005) J. Med. Chem. 48, 6090-106], we report here experimental data and theoretical analysis of their inhibitory activity toward Plm IV. All compounds share a 1,2-dihydroxyethylene unit as the transition state mimic. They possess symmetric P1 and P1' side chains and either a diacylhydrazine, a five-membered oxadiazole ring, or a retroamide at the P2 and P2' positions. Experimental binding affinities are compared to those predicted by the linear interaction energy (LIE) method and an empirical scoring function, using both a crystal structure and a homology model for the enzyme. Molecular dynamics (MD) simulations of the modeled complexes allow a rational interpretation of the structural determinants for inhibitor binding. A ligand bearing a P2 and P2' symmetric oxadiazole which is devoid of amide bonds is identified both experimentally and theoretically as the most potent inhibitor of Plm IV. For the P2 and P2' asymmetric compounds, the results are consistent with earlier predictions regarding the mode of binding of this class of inhibitors to Plm II. Theoretical estimation of selectivity for some compounds is also reported. Significant features of the Plm IV binding pocket are discussed in comparison to related enzymes, and the results obtained here should be helpful for further optimization of inhibitors.     

New benzimidazole derivatives as antiplasmodial agents and plasmepsin inhibitors: synthesis and analysis of structure-activity relationships

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ?3μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.     

Additional interaction of allophenylnorstatine-containing tripeptidomimetics with malarial aspartic protease plasmepsin II

Based on a highly potent allophenylnorstatine-containing inhibitor, KNI-10006, against the plasmepsins of Plasmodium falciparum, we synthesized a series of tripeptide-type compounds with various N-terminal moieties and evaluated their inhibitory activities against plasmepsin II. Certain phenylacetyl derivatives exhibited extremely high affinity with K(i) values of less than 0.1n M suggesting successful hydrophobic interactions.     

Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum

Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite Plasmodium falciparum, and the only one with no crystal structure reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.4 and 3.1?, respectively. The apoenzyme crystallized in the orthorhombic space group P2(1)2(1)2(1) with two molecules in the asymmetric unit and the structure has been refined to the final R-factor of 20.7%. The KNI-10006 bound enzyme crystallized in the tetragonal space group P4(3) with four molecules in the asymmetric unit and the structure has been refined to the final R-factor of 21.1%. In the PMI-KNI-10006 complex, the inhibitors were bound identically to all four enzyme molecules, with the opposite directionality of the main chain of KNI-10006 relative to the direction of the enzyme substrates. Such a mode of binding of inhibitors containing an allophenylnorstatine-dimethylthioproline insert in the P1-P1' positions, previously reported in a complex with PMIV, demonstrates the importance of satisfying the requirements for the proper positioning of the functional groups in the mechanism-based inhibitors towards the catalytic machinery of aspartic proteases, as opposed to binding driven solely by the specificity of the individual enzymes. A comparison of the structure of the PMI-KNI-10006 complex with the structures of other vacuolar plasmepsins identified the important differences between them and may help in the design of specific inhibitors targeting the individual enzymes.     

Design of inhibitors against HIV, HTLV-I, and Plasmodium falciparum aspartic proteases

Aspartic proteases have emerged as targets for substrate-based inhibitor design due to their vital roles in the life cycles of the organisms that cause AIDS, malaria, leukemia, and other infectious diseases. Based on the concept of mimicking the substrate transition-state, we designed and synthesized a novel class of aspartic protease inhibitors containing the hydroxymethylcarbonyl (HMC) isostere. An unnatural amino acid, allophenylnorstatine [Apns; (2 S ,3 S )-3-amino-2-hydroxy-4-phenylbutyric acid], was incorporated at the P1 site in a series of peptidomimetic compounds that mimic the natural substrates of the HIV, HTLV-I, and malarial aspartic proteases. From extensive structure-activity relationship studies, we were able to identify a series of highly potent peptidomimetic inhibitors of HIV protease. One highly potent inhibitor of the malarial aspartic protease (plasmepsin II) was identified. Finally, a promising lead compound against the HTLV-I protease was identified.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.     

The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 x 10(10) M(-1) or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (Delta G = -RTlnK(a)). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (-7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.     

Novel uncomplexed and complexed structures of plasmepsin II,an aspartic protease from Plasmodium falciparum

Malaria remains a human disease of global significance and a major cause of high infant mortality in endemic nations. Parasites of the genus Plasmodium cause the disease by degrading human hemoglobin as a source of amino acids for their growth and maturation. Hemoglobin degradation is initiated by aspartic proteases, termed plasmepsins, with a cleavage at the alpha-chain between residues Phe33 and Leu34. Plasmepsin II is one of the four catalytically active plasmepsins that has been identified in the food vacuole of Plasmodium falciparum. Novel crystal structures of uncomplexed plasmepsin II as well as the complex with a potent inhibitor have been refined with data extending to resolution limits of 1.9A and 2.7A, and to R factors of 17% and 18%, respectively. The inhibitor, N-(3-[(2-benzo[1,3]dioxol-5-yl-ethyl)[3-(1-methyl-3-oxo-1,3-dihydro-isoindol-2-yl)-propionyl]-amino]-1-benzyl-2-(hydroxypropyl)-4-benzyloxy-3,5-dimethoxy-benzamide, belongs to a family of potent non-peptidic inhibitors that have large P1' groups. Such inhibitors could not be modeled into the binding cavity of the structure of plasmepsin II in complex with pepstatin A. Our structures reveal that the binding cavities of the new complex and uncomplexed plasmepsin II are considerably more open than that of the pepstatin A complex, allowing for larger heterocyclic groups in the P1', P2' and P2 positions. Both complexed and uncomplexed plasmepsin II crystallized in space group P2, with one monomer in the asymmetric unit. The structures show extensive interlocking of monomers around the crystallographic axis of symmetry, with areas in excess of 2300A(2) buried at the interface, and a loop of one monomer interacting with the binding cavity of the 2-fold related monomer. Electron density for this loop is only fully ordered in the complexed structure.     

Parasite killing in Plasmodium vivax malaria by nitric oxide: implication of aspartic protease inhibition

Nitric oxide (NO) is known to possess antiparasitic activity towards Plasmodium species. Parasite proteases are currently considered to be promising targets for antimalarial chemotherapy. In the present study, we have studied the inhibitory effect of NO on the activity of plasmepsin in Plasmodium vivax, the pepsin-like aspartic protease which is believed to be involved in the cleavage during hemoglobin degradation in Plasmodium falciparum. NO donors (+/-) (E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) were found to inhibit this plasmepsin activity in a dose-dependent manner in purified P. vivax aspartic protease enzyme extracts. This inhibitory effect may be attributable to the nitrosylation of the cysteine residue at the catalytic site. However, an inhibitor of aspartic protease activity, namely pepstatin, was also found to inhibit (IC50 3 microM ) the enzyme activity, which we have used as a positive control. Our results therefore provide novel insights into the pathophysiological mechanisms, and will be useful for designing strategies for selectively upregulating NO production in P. vivax infections for antimalarial chemotherapy and also biochemical adaptations of the malaria parasite for survival in the host erythrocytes with a better understanding of the protease substrate interactions.