Karen L. White

A Comprehensive Survey of Sequence Variation in the ABCA4 (ABCR) Gene in Stargardt Disease and Age-Related Macular Degeneration

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria

Ozonide OZ439 is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. OZ439 has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients. Herein, we describe the discovery of OZ439 and the exceptional antimalarial and pharmacokinetic properties that led to its selection as a clinical drug development candidate. In vitro, OZ439 is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC50 values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, OZ439 completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, OZ439 exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. The outstanding efficacy and prolonged blood concentrations of OZ439 are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death.

Quinolone-3-Diarylethers: A New Class of Antimalarial Drug

ELQ-300, an investigational drug for treating and preventing malaria, shows potent transmission-blocking activity in rodent models of malaria. Taking the Bite Out of Malaria Malaria is spread from person to person by mosquitoes that inject 8 to 10 sporozoite forms of the parasite in a single bite. The sporozoites reproduce in the liver to produce 10,000 to 30,000 merozoites before the liver schizont ruptures and parasites flood into the bloodstream where the absolute parasite burden may increase to a thousand billion (1012) circulating parasites. Some of these parasites develop into gametocytes that may be ingested by another mosquito where they progress through ookinete, oocyst, and sporozoite stages to complete the cycle. Like quinine, most antimalarial drugs in use today target only the symptomatic blood stage. The efficacy of these drugs has been compromised by resistance, and so there is a pressing need for new drugs that target multiple stages of the parasite life cycle for use in malaria treatment and prevention. Clearly, it is advantageous to strike at the liver stage where parasite numbers are low, to diminish the likelihood of selecting for a resistant mutant and before the infection has a chance to weaken the defenses of the human host. In a new study, Nilsen and colleagues describe ELQ-300, a 4(1H)-quinolone-3-diarylether, which targets the liver and blood stages, including the forms that are crucial to disease transmission (gametocytes, zygotes, and ookinetes). In mouse models of malaria, a single oral dose of 0.03 mg/kg prevented sporozoite-induced infections, whereas four daily doses of 1 mg/kg achieved complete cures of patent infections. ELQ-300 is a preclinical candidate that may be coformulated with other antimalarials to prevent and treat malaria, with the potential to aid in eradication of the disease. The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite’s life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite’s mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

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.

Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h, the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1h−/Y male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1h−/Y mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse.

Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with antimalarial activity in mice.

Plasmodium falciparum causes 1-2 million deaths annually. Yet current drug therapies are compromised by resistance. We previously described potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase (PfDHODH) that inhibited parasite growth in vitro; however, they showed no activity in vivo. Here we show that lack of efficacy against P. berghei in mice resulted from a combination of poor plasma exposure and reduced potency against P. berghei DHODH. For compounds containing naphthyl (DSM1) or anthracenyl (DSM2), plasma exposure was reduced upon repeated dosing. Phenyl-substituted triazolopyrimidines were synthesized leading to identification of analogs with low predicted metabolism in human liver microsomes and which showed prolonged exposure in mice. Compound 21 (DSM74), containing p-trifluoromethylphenyl, suppressed growth of P. berghei in mice after oral administration. This study provides the first proof of concept that DHODH inhibitors can suppress Plasmodium growth in vivo, validating DHODH as a new target for antimalarial chemotherapy.

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.

Malarial dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target

Malarial dihydrofolate reductase (DHFR) is the target of antifolate antimalarial drugs such as pyrimethamine and cycloguanil, the clinical efficacy of which have been compromised by resistance arising through mutations at various sites on the enzyme. Here, we describe the use of cocrystal structures with inhibitors and substrates, along with efficacy and pharmacokinetic profiling for the design, characterization, and preclinical development of a selective, highly efficacious, and orally available antimalarial drug candidate that potently inhibits both wild-type and clinically relevant mutated forms of Plasmodium falciparum (Pf) DHFR. Important structural characteristics of P218 include pyrimidine side-chain flexibility and a carboxylate group that makes charge-mediated hydrogen bonds with conserved Arg122 (PfDHFR-TS amino acid numbering). An analogous interaction of P218 with human DHFR is disfavored because of three species-dependent amino acid substitutions in the vicinity of the conserved Arg. Thus, P218 binds to the active site of PfDHFR in a substantially different fashion from the human enzyme, which is the basis for its high selectivity. Unlike pyrimethamine, P218 binds both wild-type and mutant PfDHFR in a slow-on/slow-off tight-binding mode, which prolongs the target residence time. P218, when bound to PfDHFR-TS, resides almost entirely within the envelope mapped out by the dihydrofolate substrate, which may make it less susceptible to resistance mutations. The high in vivo efficacy in a SCID mouse model of P. falciparum malaria, good oral bioavailability, favorable enzyme selectivity, and good safety characteristics of P218 make it a potential candidate for further development.

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.

VMD2 mutations in vitelliform macular dystrophy (Best disease) and other maculopathies

Mutations in the gene VMD2 are associated with autosomal dominant vitelliform macular dystrophy (Best disease). VMD2 is expressed in the retinal pigment epithelium and codes for a 585 amino acid putative transmembrane protein with undetermined functional properties. To date, 48 different mutations, predominantly missense, have been described in Best disease families. These mutations generally affect amino acids in the first 50% of the protein, and occur in four distinct clusters possibly representing regions of functional importance. VMD2 has also been investigated in other macular diseases. Mutations have been documented in a significant percentage of patients with adult vitelliform macular dystrophy (AVMD) and in a single case of “bulls‐eye” maculopathy. Results of analysis in two large series of individuals with age‐related macular degeneration (AMD) suggest that VMD2 does not play a major role in this prevalent disorder. Hum Mutat 15:301–308, 2000.