A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

Inhibitors of ApiAP2 protein DNA binding exhibit multistage activity against Plasmodium parasites.

Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified putative AP2-EXP interacting compounds. Four compounds were found to block DNA binding by AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that interact with AP2 DNA binding domains. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

Antiplasmodial peptaibols act through membrane directed mechanisms.

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Genome-wide association study of aspirin-exacerbated respiratory disease in a Korean population.

Aspirin-exacerbated respiratory disease (AERD) is a nonallergic clinical syndrome characterized by a severe decline in forced expiratory volume in one second (FEV1) following the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin. The effects of genetic variants have not fully explained all of the observed individual differences to an aspirin challenge despite previous attempts to identify AERD-related genes. In the present study, we performed genome-wide association study (GWAS) and targeted association study in Korean asthmatics to identify new genetic factors associated with AERD. A total of 685 asthmatic patients without AERD and 117 subjects with AERD were used for the GWAS of the first stage, and 996 asthmatics without AERD and 142 subjects with AERD were used for a follow-up study. A total of 702 SNPs were genotyped using the GoldenGate assay with the VeraCode microbead. GWAS revealed the top-ranked variants in 3' regions of the HLA-DPB1 gene. To investigate the detailed genetic effects of an associated region with the risk of AERD, a follow-up targeted association study with the 702 single nucleotide polymorphisms (SNPs) of 14 genes was performed on 802 Korean subjects. In a case-control analysis, HLA-DPB1 rs1042151 (Met105Val) shows the most significant association with the susceptibility of AERD (p = 5.11 × 10(-7); OR = 2.40). Moreover, rs1042151 also shows a gene dose for the percent decline of FEV1 after an aspirin challenge (p = 2.82 × 10(-7)). Our findings show that the HLA-DPB1 gene polymorphism may be the most susceptible genetic factor for the risk of AERD in Korean asthmatics and confirm the importance of HLA-DPB1 in the genetic etiology of AERD.

Advances in malaria pharmacology and the online guide to MALARIA PHARMACOLOGY: IUPHAR review 38.

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome.

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation.

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.

The genetic effect of copy number variations on the risk of alcoholism in a Korean population.

BACKGROUND: Alcoholism, a chronic behavioral disorder characterized by excessive alcohol consumption, has been a leading cause of morbidity and premature death. This condition is believed to be influenced by genetic factors. As copy number variation (CNV) has been recently discovered in human genome, genomic diversity of human genome is more frequent than previously thought. Many studies have reported evidences that CNV is associated with the development of complex diseases. In this study, we hypothesized that CNV can predict the risk of alcoholism. METHODS: Using the Illumina HumanHap660W-Quad BeadChip (∼660 k markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 116 alcoholic cases and 1,022 healthy controls (total n = 1,138) in a Korean population. To identify alcoholism-associated CNV regions, we performed a genome-wide association analysis, using multivariate logistic regression model controlling for age and gender. RESULTS: We identified a total of 255,732 individual CNVs and 3,261 CNV regions (1,067 common CNV regions, frequency > 1%) in this study. Results from multivariate logistic regression showed that the chr20:61195302-61195978 regions were significantly associated with the risk of alcoholism after multiple corrections (p = 5.02E-05, p(corr) = 0.04). Most of the identified variations in this study overlapped with the previously reported CNVs in the Database of Genomic Variants (95.3%). The identified CNVs, which encompassed 3,226 functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the cell part, in developmental processes, in cell communication, in neurological system process, in sensory perception of smell and chemical stimulus, and in olfactory receptor activity. CONCLUSIONS: This is the first genome-wide association study to investigate the relationship between common CNV and alcoholism. Our results suggest that the newly identified CNV regions may contribute to the development of alcoholism.

Genetic variations in KIFC1 and the risk of aspirin exacerbated respiratory disease in a Korean population: an association analysis.

Modest effects of genes in various pathways are significant in the etiology of complex human diseases, including aspirin exacerbated respiratory disease (AERD). By functioning as a relevant component of respiratory processes, the human kinesin family member C1 (KIFC1) is hypothesized to play a role in AERD pathogenesis. A case-control analysis was carried out by comparing the genotype distribution of six KIFC1 single-nucleotide polymorphisms between 93 AERD cases and 96 aspirin-tolerant asthma controls in a Korean population. After controlling for confounds, logistic and regression models via various modes of genetic inheritance facilitated the association analysis. Initial results revealed significant association at 0.05 level of significance between several KIFC1 variations and AERD (P = 0.01-0.05, OR = 1.81-1.90) as well as fall rate of forced expiratory volume in the 1st second, an important diagnostic marker of airways constriction (P = 0.04-0.05). However, the signals were not deemed significant after multiple testing corrections (P (corr) > 0.05). Although the results do not support a major role of KIFC1 in AERD pathogenesis in a Korean asthma cohort, further replication and validation studies are required to clarify the current findings.

Association of FANCC polymorphisms with FEV1 decline in aspirin exacerbated respiratory disease.

Aspirin exacerbated respiratory disease (AERD) is a clinical condition characterized by severe decline in forced expiratory volume in one second (FEV1) following the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin. The exacerbated inflammatory response in Fancc-deficient mice has been reported to be associated with hemopoietic responses that are also related to AERD pathogenesis. To investigate associations of FANCC polymorphisms with AERD and related phenotypes, this study genotyped 25 common single nucleotide polymorphisms (SNPs) in a total of 592 Korean asthmatics including 163 AERD and 429 aspirin-tolerant asthma (ATA) subjects. Logistic analysis revealed that genetic polymorphisms of the FANCC gene might not be directly related to AERD development and nasal polyposis (P > 0.05). However, the FEV1 decline by aspirin provocation showed significant associations with FANCC polymorphisms (P = 0.006-0.04) and a haplotype (unique to rs4647416G > A, P = 0.01 under co-dominant, P = 0.006 under recessive model). In silico analysis showed that the "A" allele of rs4647376C > A, which was more prevalent in AERD than in ATA, could act as a potential branch point (BP) site for alternative splicing (BP score = 4.16). Although replications in independent cohorts and further functional evaluations are still needed, our preliminary findings suggest that FANCC polymorphisms might be associated with the obstructive symptoms in allergic diseases.

Potential association of DDR1 genetic variant with FEV1 decline by aspirin provocation in asthmatics.

BACKGROUND: The discoidin domain receptor tyrosine kinase 1 (DDR1) is positioned within the major histocompatibility complex (MHC) region which plays an important role in the immune system. In addition, DDR1 has been elucidated to be downregulated during the epithelial-mesenchymal transition of bronchial epithelium. OBJECTIVE: To investigate the potential genetic associations between DDR1 and aspirin-exacerbated respiratory disease (AERD), this study conducted association studies of DDR1 single nucleotide polymorphisms (SNPs) with AERD and the obstructive symptom of forced expiratory volume in 1 s (FEV(1)) decline after aspirin provocation. METHODS: Nine common SNPs were genotyped in 93 AERD patients and 96 aspirin-tolerant asthma (ATA) controls. The genotype distributions of all loci were in Hardy-Weinberg equilibrium (HWE; p > .05). Results. In the results of logistic analyses using age, sex, smoking status, and atopy as covariates, DDR1 rs1264320 in the intronic region showed a potent association signal with FEV(1) decline by aspirin provocation in asthmatics of this study even after corrections for multiple testing (p = .003 and corrected p = .01). However, the variants of DDR1 were not significantly associated with the AERD development (corrected p > .05). On further comparison of FEV(1) decline by aspirin provocation between AERD and ATA, the variant rs1264320 was found to be associated with the FEV(1) decline of ATA rather than AERD. CONCLUSION: Despite the need for further functional evaluations and replications, we conclude that DDR1 polymorphisms are not likely to contribute to predispositions of AERD, but may be potentially associated with FEV(1) decline by aspirin provocation in asthmatics.

DCBLD2 gene variations correlate with nasal polyposis in Korean asthma patients.

BACKGROUND: Nasal polyps are abnormal lesions that cause airway obstruction and can occur along with other respiratory diseases. On account of its association with aspirin exacerbated respiratory disease (AERD), the human discoidin, CUB and LCCL domain containing 2 (DCBLD2) is hypothesized to be a candidate gene for the development of nasal polyps in asthma patients. METHODS: A total of 12 single-nucleotide polymorphisms (SNPs) were genotyped in 467 Korean asthma patients who were stratified further into 108 AERD and 353 aspirin-tolerant asthma (ATA) subgroups. Five major haplotypes were inferred from pairwise comparison of the polymorphisms. The patients were matched to control for confounds, and differences in the frequency distribution of DCBLD2 SNPs and haplotypes were analyzed using logistic models via various modes of genetic inheritance. RESULTS: Results reveal significant association of rs828618 and DCBLD2_ht1 with nasal polyposis in the overall asthma patients group (P = 0.006, P(corr) = 0.05). Interestingly, the strength of association were maintained in the ATA subgroup (P = 0.007, P(corr) = 0.06), and moderate correlation was detected in the AERD subgroup (P = 0.04-0.05, P(corr) > 0.05). CONCLUSIONS: Although further replication and validation are needed, these findings suggest that DCBLD2 could be a potential marker and drug target for treatment of nasal polyposis in Korean asthma patients.

Effect of diffuse panbronchiolitis critical region 1 polymorphisms on the risk of aspirin-exacerbated respiratory disease in Korean asthmatics.

BACKGROUND: The functional role of the human diffuse panbronchiolitis critical region 1 (DPCR1) gene, located in the major histocompatibility complex class I, has not been widely investigated. However, this gene is a well known genetic marker for diffuse panbronchiolitis, a disease affecting human respiratory bronchioles. In this study we explored the association between polymorphisms in DPCR1 and aspirin-exacerbated respiratory disease (AERD), an asthma phenotype. METHODS: Genotyping of 6 polymorphisms was carried out in a total of 189 Korean asthmatic patients stratified into 93 AERD cases and 96 aspirin tolerant asthma controls. Subjects who exhibited significant decrease of FEV(1) by aspirin provocation were identified as AERD subjects. Logistic and regression analyses were performed to investigate the association between DPCR1 polymorphisms and the risk of AERD as well as FEV(1) decline. RESULTS: Initial analysis revealed significant association of rs2517449 with AERD, with a P value of .03 via a recessive model; however, the association signal disappeared after multiple testing corrections. In addition, rs2517449 and rs2240804 also showed association signals with decline of FEV(1) after aspirin provocation (P = .007 and .03, respectively, in a recessive model). After testing for multiple comparisons, only the association signal from rs2517449 was retained (P(corr) = .04), while other polymorphisms showed no associations with the risk of AERD and FEV(1) decline. CONCLUSIONS: Our results show that polymorphisms in DPCR1 are not associated with the risk of AERD.

Functional genomics of RAP proteins and their role in mitoribosome regulation in Plasmodium falciparum.

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

The Plasmodium falciparum ABC transporter ABCI3 confers parasite strain-dependent pleiotropic antimalarial drug resistance.

Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.