Xiaoyi Deng

Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential antimalarials, we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound toward clinical candidate status.

A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

The antimalarial drug DSM265 displays activity against blood and liver stages of Plasmodium falciparum and has a long predicted half-life in humans. Long-acting new treatment for drug-resistant malaria Malaria kills 0.6 million people annually, yet current malaria drugs are no longer fully effective because the parasite that causes malaria is becoming resistant to these agents. Phillips et al. have identified a new drug that kills both drug-sensitive and drug-resistant malaria parasites by targeting the ability of the parasite to synthesize the nucleotide precursors required for synthesis of DNA and RNA. This drug kills parasites in both the blood and liver and is sufficiently long-acting that it is expected to cure malaria after a single dose or to be effective if dosed weekly for chemoprevention. Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.

Lead-optimization of aryl and aralkyl amine based triazolopyrimidine inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice

Malaria is one of the leading causes of severe infectious disease worldwide; yet, our ability to maintain effective therapy to combat the illness is continually challenged by the emergence of drug resistance. We previously reported identification of a new class of triazolopyrimidine-based Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors with antimalarial activity, leading to the discovery of a new lead series and novel target for drug development. Active compounds from the series contained a triazolopyrimidine ring attached to an aromatic group through a bridging nitrogen atom. Herein, we describe systematic efforts to optimize the aromatic functionality with the goal of improving potency and in vivo properties of compounds from the series. These studies led to the identification of two new substituted aniline moieties (4-SF(5)-Ph and 3,5-Di-F-4-CF(3)-Ph), which, when coupled to the triazolopyrimidine ring, showed good plasma exposure and better efficacy in the Plasmodium berghei mouse model of the disease than previously reported compounds from the series.

Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED50 values in the 4-day murine P. berghei efficacy model of 13–21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.

Fluorine Modulates Species Selectivity in the Triazolopyrimidine Class of Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors

Malaria is one of the most serious global infectious diseases. The pyrimidine biosynthetic enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is an important target for antimalarial chemotherapy. We describe a detailed analysis of protein–ligand interactions between DHODH and a triazolopyrimidine-based inhibitor series to explore the effects of fluorine on affinity and species selectivity. We show that increasing fluorination dramatically increases binding to mammalian DHODHs, leading to a loss of species selectivity. Triazolopyrimidines bind Plasmodium and mammalian DHODHs in overlapping but distinct binding sites. Key hydrogen-bond and stacking interactions underlying strong binding to PfDHODH are absent in the mammalian enzymes. Increasing fluorine substitution leads to an increase in the entropic contribution to binding, suggesting that strong binding to mammalian DHODH is a consequence of an enhanced hydrophobic effect upon binding to an apolar pocket. We conclude that hydrophobic interactions between fluorine and hydrocarbons provide significant binding energy to protein–ligand interactions. Our studies define the requirements for species-selective binding to PfDHODH and show that the triazolopyrimidine scaffold can alternatively be tuned to inhibit human DHODH, an important target for autoimmune diseases.

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

Altering pyrimidine levels in influenza virus– or VSV-infected cells reverses the inhibitory effect of viral proteins NS1 and M on host mRNA nuclear export, restoring expression of host antiviral factors.

Tetrahydro-2-naphthyl and 2-Indanyl Triazolopyrimidines Targeting Plasmodium falciparum Dihydroorotate Dehydrogenase Display Potent and Selective Antimalarial Activity

Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.

Evolution of Substrate Specificity within a Diverse Family of β/α-Barrel-fold Basic Amino Acid Decarboxylases X-RAY STRUCTURE DETERMINATION OF ENZYMES WITH SPECIFICITY FOR l-ARGININE AND CARBOXYNORSPERMIDINE

Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu314, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

The X-ray structure of Plasmodium falciparum dihydroorotate dehydrogenase bound to a potent and selective N-phenylbenzamide inhibitor reveals novel binding-site interactions

Plasmodium species are protozoan parasites that are the causative agent of malaria. Malaria is a devastating disease, and its treatment and control have been hampered by the propensity of the parasite to become drug-resistant. Dihydroorotate dehydrogenase (DHODH) has been identified as a promising new target for the development of antimalarial agents. Here, the X-ray structure of P. falciparum DHODH bound to a potent and selective N-phenylbenzamide-based inhibitor (DSM59) is described at 2.3 Å resolution. The structure elucidates novel binding-site interactions and shows how conformational flexibility of the enzyme leads to the ability to bind diverse chemical structures with high affinity. This information provides new insight into the design of high-affinity DHODH inhibitors for the treatment of malaria.