Malaria is an infectious disease caused by parasites of the genus

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization.

N-Myristoyltransferase (NMT) is an attractive antiprotozoan drug target. A lead-hopping approach was utilized in the design and synthesis of novel benzo[b]thiophene-containing inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) NMT. These inhibitors are selective against Homo sapiens NMT1 (HsNMT), have excellent ligand efficiency (LE), and display antiparasitic activity in vitro. The binding mode of this series was determined by crystallography and shows a novel binding mode for the benzothiophene ring.

Caging of a widely used Von Hippel Lindau E3 ligase ligand for targeted protein degradation with PROTACs allows light-activated proteolysis.

SUMMARY 12 13Infections caused by protozoan parasites are among the most widespread and intractable transmissible 14 diseases affecting the developing world, with malaria and leishmaniasis being most costly in terms of 15 morbidity and mortality. Although new drugs are urgently required against both diseases in the face of 16 ever-rising resistance to frontline therapies, very few candidates passing through development 17 pipelines possess a known and novel mode of action. Set in the context of drugs currently in use and 18 under development, we present the evidence for N-myristoyltransferase (NMT), an enzyme that N-19 terminally lipidates a wide range of specific target proteins through post-translational modification, as 20 a potential drug target in malaria and the leishmaniases. We discuss the limitations of current 21 knowledge regarding the downstream targets of this enzyme in protozoa, and our recent progress 22 towards potent cell-active NMT inhibitors against the most clinically-relevant species of parasite. 23Finally, we outline the next steps required in terms of both tools to understand N-myristoylation in 24 protozoan parasites, and the generation of potential development candidates based on the output of 25 our recently-reported high-throughput screens. 26 INTRODUCTION 27Malaria 28 Malaria is a disease caused by infection of a human host with protozoan parasites of the genus 29Plasmodium, and is a devastating global health issue with approximately 200 million cases and 30 1 million deaths in 2010 alone (Murray et al. 2012). The complex life cycle of malaria parasites 31 spreads across two hosts and five host tissues whilst undergoing at least ten distinct morphological 32 transitions (Sturm et al. 2006; Mackinnon and Marsh 2010). Replication of parasites and subsequent 33 rupture of erythrocytes in the intra-erythrocytic stages are responsible for the clinical symptoms of 34 malaria, and the majority of drugs target these asexual (human-host) stages of the life cycle. Some 35 species of malaria, most notably Plasmodium vivax, can exist in a latent liver hypnozoite form that 36 can cause relapse even after clearance of bloodstream parasites (Derbyshire et al. 2012; Rodrigues et 37 al. 2012). Of the five relevant species of human parasite, the vast majority of deaths occur from P. 38 falciparum infections, which is the typical cause of severe malaria (Claessens et al. 2012). This has 39 led to the majority of drug discovery efforts focussing on P. falciparum, typically at the expense of 40 other species. Although the demand for new P. falciparum drugs is in no doubt, P. vivax is 41 responsible for the majority of worldwide malaria endemicity (Price et al. 2009; WHO 2011). 42However, difficulties culturing the parasite (Udomsangpetch et al. 2007) For the latter half of the 20 th century, antimalarial drug discovery was a success story for natural 47 product-inspired therapies, by far the most widely used of which are chloroquine (Loeb et al. 1946) 48 and artemisinin (Miller and Su 2011). 49Chlo...

Truncation converted a Plasmodium N-myristoyltransferase inhibitor into a Leishmania-selective series, leading to a potent L. donovani NMT inhibitor through structure-guided design.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.