Kevin D. Read

Automated design of ligands to polypharmacological profiles

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand–target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

Central Nervous System Drug Disposition: The Relationship between in Situ Brain Permeability and Brain Free Fraction

The dispositions of 50 marketed central nervous system (CNS) drugs into the brain have been examined in terms of their rat in situ (P) and in vitro apparent membrane permeability (Papp) alongside lipophilicity and free fraction in rat brain tissue. The inter-relationship between these parameters highlights that both permeability and brain tissue binding influence the uptake of drugs into the CNS. Hydrophilic compounds characterized by low brain tissue binding display a strong correlation (R2 = 0.82) between P and Papp, whereas the uptake of more lipophilic compounds seems to be influenced by both Papp and brain free fraction. A nonlinear relationship is observed between logPoct and P over the 6 orders of magnitude range in lipophilicity studied. These findings corroborate recent reports in the literature that brain penetration is a function of both rate and extent of drug uptake into the CNS.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for ∼30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target—T. brucei N-myristoyltransferase—leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Receptor Occupancy and Brain Free Fraction

This study was designed to investigate whether brain unbound concentration (Cu,brain) is a better predictor of dopamine D2 receptor occupancy than total brain concentration, cerebrospinal fluid concentration (CCSF), or blood unbound concentration (Cu,blood). The ex vivo D2 receptor occupancy and concentration-time profiles in cerebrospinal fluid, blood, and brain of six marketed antipsychotic drugs were determined after oral administration in rats at a range of dose levels. The Cu,brain was estimated from the product of total brain concentration and unbound fraction, which was determined using a brain homogenate method. In conclusion, the Cu,brain of selected antipsychotic agents is a good predictor of D2 receptor occupancy in rats. Furthermore, Cu,brain seems to provide a better prediction of D2 receptor occupancy than CCSF or Cu,blood for those compounds whose mechanism of entry into brain tissue is influenced by factors other than simple passive diffusion.

The Anti-Trypanosome Drug Fexinidazole Shows Potential for Treating Visceral Leishmaniasis

Fexinidazole, a drug in clinical testing for African sleeping sickness, shows potential as an oral treatment for another neglected tropical disease. A New Job for an Old Drug Fever, fatigue, weight loss, and swelling of the spleen and liver are all symptoms of visceral leishmaniasis—a tropical disease that is also known as kala-azar or black fever. Caused by the protozoan parasite Leishmania donovani, which is transmitted to people through the bite of a sand fly, the disease is almost always fatal if untreated. Although several drugs exist, they are costly and not always safe, effective, or easy to administer. To address the need for better drugs, Wyllie et al. investigated the possibility of using fexinidazole to treat visceral leishmaniasis. This antiparasitic compound, developed decades ago, is now undergoing early clinical trials as an oral therapy for African sleeping sickness, a disease that is caused by a related protozoan parasite called Trypanosoma brucei. Fexinidazole’s mode of action is thought to involve a trypanosome nitroreductase; the finding that a closely related enzyme is encoded by the leishmania genome inspired Wyllie et al. to pursue fexinidazole as a therapy for visceral leishmaniasis. They found that the compound and two of its metabolites (which rapidly form in vivo) showed activity against both developmental stages of L. donovani in vitro. The metabolites were cytotoxic, killing all the parasites within 30 hours. For unclear reasons, only the metabolites were active against L. donovani grown in macrophages (the cells in which the parasite reproduces during infection). In a mouse model of visceral leishmaniasis, a daily oral dose of fexinidazole for 5 days almost completely suppressed infection—an activity that is comparable to that of drugs currently in clinical use against this deadly tropical disease. Visceral leishmaniasis kills more people than any other parasitic disease except malaria. The clinical trials of fexinidazole for African sleeping sickness have already shown that the drug is extremely safe. The discovery that it may also be a viable oral treatment for visceral leishmaniasis bodes well for those afflicted with this disease. Safer and more effective oral drugs are required to treat visceral leishmaniasis, a parasitic disease that kills 50,000 to 60,000 people each year in parts of Asia, Africa, and Latin America. Here, we report that fexinidazole, a drug currently in phase 1 clinical trials for treating African trypanosomiasis, shows promise for treating visceral leishmaniasis. This 2-substituted 5-nitroimidazole drug is rapidly oxidized in vivo in mice, dogs, and humans to sulfoxide and sulfone metabolites. Both metabolites of fexinidazole were active against Leishmania donovani amastigotes grown in macrophages, whereas the parent compound was inactive. Pharmacokinetic studies with fexinidazole (200 mg/kg) showed that fexinidazole sulfone achieves blood concentrations in mice above the EC99 (effective concentration inhibiting growth by 99%) value for at least 24 hours after a single oral dose. A once-daily regimen for 5 days at this dose resulted in a 98.4% suppression of infection in a mouse model of visceral leishmaniasis, equivalent to that seen with the drugs miltefosine and Pentostam, which are currently used clinically to treat this tropical disease. In African trypanosomes, the mode of action of nitro drugs involves reductive activation via a NADH (reduced form of nicotinamide adenine dinucleotide)–dependent bacterial-like nitroreductase. Overexpression of the leishmanial homolog of this nitroreductase in L. donovani increased sensitivity to fexinidazole by 19-fold, indicating that a similar mechanism is involved in both parasites. These findings illustrate the potential of fexinidazole as an oral drug therapy for treating visceral leishmaniasis.

Discovery of a novel class of orally active trypanocidal N-myristoyltransferase inhibitors.

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.

Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Assessing brain free fraction in early drug discovery.

Importance of the field: The incorporation of brain tissue binding routinely in CNS drug discovery screening strategies has markedly changed the way CNS drug discovery is performed and is proving to be a valuable tool in identifying new therapies for CNS diseases. For many years emphasis has been placed on the magnitude of the brain to blood ratio, the bigger the better, even though, in many cases, brain total concentration (Cbrain) has no or, at best, poor correlation with receptor occupancy/pharmacodynamic readout. Today, Cbrain values measured during in vivo experiments are corrected for the fraction unbound measured through in vitro experiments using brain tissue homogenate or brain tissue slice to obtain an estimate of the brain unbound concentration (Cu,brain), and this has been demonstrated across a range of CNS targets to give a much better correlation with receptor occupancy/pharmacodynamic readout. This apparently simple change in CNS lead optimisation strategy has de facto revolutionised the vision of the brain penetration concepts. Areas covered in this review: This review will provide an overview of the use and applications of assessing brain free fraction to determine Cu,brain. Take home message: Assessing brain free fraction to determine Cu,brain in CNS lead optimisation strategies is the surrogate of choice for rapidly assessing biophase concentration for the majority of CNS targets.

Target Validation: Linking Target and Chemical Properties to Desired Product Profile

The discovery of drugs is a lengthy, high-risk and expensive business taking at least 12 years and is estimated to cost upwards of US

Combining PET biodistribution and equilibrium dialysis assays to assess the free brain concentration and BBB transport of CNS drugs.

800 million for each drug to be successfully approved for clinical use. Much of this cost is driven by the late phase clinical trials and therefore the ability to terminate early those projects destined to fail is paramount to prevent unwanted costs and wasted effort. Although neglected diseases drug discovery is driven more by unmet medical need rather than financial considerations, the need to minimise wasted money and resources is even more vital in this under-funded area. To ensure any drug discovery project is addressing the requirements of the patients and health care providers and delivering a benefit over existing therapies, the ideal attributes of a novel drug needs to be pre-defined by a set of criteria called a target product profile. Using a target product profile the drug discovery process, clinical study design, and compound characteristics can be defined all the way back through to the suitability or druggability of the intended biochemical target. Assessment and prioritisation of the most promising targets for entry into screening programmes is crucial for maximising chances of success.