Toxicology Research, 2021, 10, 203–213

doi: 10.1093/toxres/tfaa106 Advance Access Publication Date: 15 February 2021 Review

REVIEW

A target safety assessment of the potential Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 toxicological risks of targeting plasmepsin IX/X for the treatment of Jane Barber,1 Phumzile Sikakana,1 Claire Sadler,1 Delphine Baud,2 ∗ Jean-Pierre Valentin3 and Ruth Roberts1,4, 1ApconiX, Alderley Park, Alderley Edge, SK10 4TG, UK, 2Medicines for Malaria Venture, 20 Route de Pré-Bois, Geneva 1215, Switzerland, 3UCB Biopharma SRL, Building R9, Chemin du Foriest, 1420 Braine-l’Alleud, Belgium and 4Biosciences, University of Birmingham, Edgbaston, B15 2TT, UK

∗ Correspondence address. ApconiX, Alderley Park, Alderley Edge, SK10 4DG, UK. Tel: +44 77 33 01 43 96; E-mail: [email protected]

Abstract The aspartic plasmepsin IX/X are important antimalarial drug targets due to their specificity to the malaria parasite and their vital role as mediators of disease progression. Focusing on parasite-specific targets where no human homologue exists reduces the possibility of on-target drug toxicity. However, there is a risk of toxicity driven by inadequate selectivity for plasmepsins IX/X in Plasmodium over related mammalian aspartic proteases. Of these, CatD/E may be of most toxicological relevance as CatD is a ubiquitous lysosomal enzyme present in most cell types and CatE is found in the gut and in erythrocytes, the clinically significant site of malarial infection. Based on mammalian aspartic physiology and adverse drug reactions (ADRs) to FDA-approved human immunodeficiency virus (HIV) inhibitors, we predicted several potential toxicities including β-cell and congenital abnormalities, hypotension, hypopigmentation, hyperlipidaemia, increased infection risk and respiratory, renal, gastrointestinal, dermatological, and other epithelial tissue toxicities. These ADRs to the HIV treatments are likely to be a result of host aspartic protease inhibition due a lack of specificity for the HIV protease; plasmepsins are much more closely related to human CatD than to HIV proteinase. Plasmepsin IX/X inhibition presents an opportunity to specifically target Plasmodium as an effective antimalarial treatment, providing adequate selectivity can be obtained. Potential plasmepsin IX/X inhibitors should be assayed for inhibitory activity against the main human aspartic proteases and particularly CatD/E. An investigative rodent study conducted early in drug discovery would serve as an initial risk assessment of the potential hazards identified.

Received: 28 August 2020; Revised: 30 November 2020; Accepted: 7 December 2020

© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

203 204 Toxicology Research, 2021, Vol. 10, No. 2

Graphical Abstract Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021

Introduction mitigation strategy. Here we explore the benefit–risk profile for a potential new target in treating malaria and propose next steps Despite the increase in drug approvals in recent years, drug to evaluate risk. discovery and development remain challenging with high rates of failure [1]. In the hunt for ways to make drug discovery and Malaria: the problem development more successful, there have been several excellent analyses of why drugs fail [2, 3]. Although the reasons for failure Malaria remains an overwhelming problem, particularly in are usually complex and multifactorial, ‘safety/toxicity’ is cited Africa. In 2018, there were an estimated 228 million cases of as the predominant cause of failure [2, 3] with around 40% of malaria with an estimated 405 000 deaths from the disease these failures attributable to safety issues associated with the globally. The burden of disease is heaviest in the World Health therapeutic target itself [2]. This is not surprising since many tar- Organization (WHO) African Region where 93% of all malaria gets that are attractive in treating disease are also likely to have deaths occurred. Plasmodium falciparum (P. falciparum) is the important roles in normal biology and their modulation could most prevalent malaria parasite (99.7%) in the WHO African lead to unintended consequence. So, would not it make sense to Region carried by the Anopheles mosquito, whereas P. vivax is the maximize our understanding of the biology of a potential drug predominant parasite (75%) in the WHO Region of the Americas. target in order to predict and manage issues before they arise? Children aged under 5 years old and pregnant women are in the For many projects, the drug target may be expressed in tissues most vulnerable groups affected by malaria; children aged under other than the intended therapeutic target; a careful analysis 5 account for >60% of malaria deaths in 2018 worldwide [4]. of expression profiles can help predict likely consequences of Classical uncomplicated malaria presents with a combination this and offer an insight into the benefit–risk profile. For most of symptoms including fever, chills, sweats, headaches, body anti-infectives (antibiotics, antifungals, antibacterials, antiproto- aches, nausea, and vomiting. Severe malarial infections are zoals), the target may be specific to the infectious agent suggest- characterized by serious organ failures or abnormalities in the ing that the potential for on-target toxicity is limited. However, patient’s blood or metabolism [5]. The current fight against this is not always the case since there may be related mammalian the disease is being waged on a variety of fronts, including targets that need to be considered to develop an overall risk the distribution of bed nets, the promotion of indoor spraying, Barber et al. 205

and the development of new medicines, vaccines, and insec- the cleavage of that are to be exported to the host cell ticides. Emerging parasite resistance to the currently available [18], and is therefore already a focus of drug development. PMIX drugs is of increasing major concern. and PMX have recently been highlighted as mediators of egress The first instance of antimalarial drug resistance was with and invasion of the parasite further exposing the parasite at its chloroquine. Chloroquine resistance in P. falciparum was devel- most vulnerable state accentuating these enzymes as potential oped independently in multiple areas of Southeast Asia, Oceania, drug targets [15, 19, 20]. and South America in the 1950/60s. Subsequently chloroquine Within the erythrocyte the Plasmodium parasite matures resistance spread to nearly all areas of the world where P. fal- from a ring to a trophozoite and then a schizont which ciparum malaria is transmitted [6]. Since then P. falciparum also produces merozoites primed for invasion (Fig. 1). The release developed resistance to nearly all of the other currently avail- of merozoites, known as egress, is a two-step process: the able antimalarial drugs, including sulfadoxine/pyrimethamine, degradation of the parasitophorous vacuole (PV), which encloses mefloquine, halofantrine, and quinine [7]. Although resistance the merozoites, and erythrocyte membrane. Invasion of a new to these drugs tends to be much less widespread geographically, erythrocyte takes only 10–30 s and involves recognition of the in some areas of the world, the impact of multidrug resistant erythrocyte membrane, attachment, reorientation, and entry Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 malaria can be extensive [8]. Most recently, resistance to the [21]. The proteins involved in invasion and egress are packaged artemisinin and non-artemisinin components of artemisinin- into several secretory organelles in the merozoite including the based combination therapy has emerged in parts of Southeast rhoptries and micronemes (invasion) and the dense granule-like Asia, impacting the efficacy of this vital antimalarial class [9]. exonemes (egress). The activity of several serine and cysteine Chloroquine-resistant P. vivax malaria has also been identified proteases promotes the destabilization of the PV and erythrocyte in a number of regions including Papua New Guinea, Southeast membranes which surround the parasite [22]. PMX processes Asia, Ethiopia, and Madagascar [6]. Therefore, there is still a subtilisin-like protease 1 (SUB1) to activate it. Inhibition of PMX continuous need for novel, differentiated approaches to treat results in the accumulation of SUB1 precursor [10]. Mature SUB1 malaria. is required for the degradation of both the PV and erythrocyte membrane to allow the dissemination of merozoites from a Plasmepsins: drug targets for malaria mother schizont. To initiate the egress cascade, SUB1 activates cysteine proteases called SERAs and merozoite surface proteins As malaria routinely develops resistance to drugs, there is a con- called MSPs [23]. Full block of PMX traps parasites within the tinual need for novel antimalarials to combat the disease. This PV membrane, whereas partial block allows egress from this is especially vital in the case of multidrug resistant P. falciparum membrane but prevents escape from the erythrocyte membrane. as the number of available therapies are reducing and becoming This suggests a higher level of activated SUB1 is required for less effective. Development of small molecule plasmepsin (PM) its effects on the erythrocyte [10]. Invasion also crucially relies IX/X inhibitors presents a unique opportunity to specifically tar- on serine proteases to activate or remove ligands involved in get the malarial aspartic protease to prevent parasitic infection. interactions with the host erythrocyte [24]. Aspartic proteases are key contributors to the pathogenicity of PMIX and PMX are expressed in mature blood-stage P. falciparum. PMs IX and X are unique to Plasmodium and have schizonts and invasive merozoites and fulfil indispensable indispensable functions in the parasite making them ideal drug functions. PMIX and PMX are specifically involved in these targets [10–13]. Focusing on parasite-specific targets for which egress-invasion processes evidenced in two recent studies no human host homologue exists reduces the chance of drug by Pino et al.[25]andNasamuet al. [11]. Both studies used toxicity. However, adequate selectivity for PMs IX/X over related similar approaches to characterize PMIX and PMX, supporting mammalian aspartic proteases is critical. If there is insufficient their validation as drug targets whilst also presenting two selectivity for the Plasmodium aspartic proteases then human novel inhibitors 49c and CWHM-117. Both groups were unable toxicity is to be expected, the nature of which will depend on the to generate parasite knockouts, inferring essential survival human aspartic proteases that are inhibited by the molecule. functions associated with PMIX/X. The groups reported that the In the erythrocyte stage of infection, intraerythrocytic malaria compounds lead to egress and invasion phenotypes similar to parasites degrade haemoglobin (Hb) to provide nutrients for their those seen in the knockdown parasites. When 49c was removed own growth and maturation. Hb degradation into its constituent before parasite egress, the parasite invaded new erythrocytes amino acids is a multistep process involving several degradative normally indicating that treatment, at least with this drug, enzymes which occurs in the acidic parasitic food vacuole (FV). is time dependent. For 49c to inhibit egress, it needs to be The FV contains aspartic proteases, cysteine proteases, and met- present for an extended period, to enable it to block the gradual alloproteases, which are all believed to play roles in an ordered accumulation of mature SUB1 to an extent sufficient to reduce Hb degradation pathway [14]. Because the degradation of Hb is SUB1 activity below the threshold required for membrane vital for parasite survival, strategies to inhibit this degradation rupture. Crucially, this impacts on the required pharmacokinetic pathway offers a valid approach by which to develop novel (PK) properties of PMIX/X inhibitors, which may require long chemotherapeutic agents [11]. In the first step of degradation, Hb plasma half-lives in vivo and/or long residence times in order is broken down into large fragments by PM enzymes [14]. to exert the required physiologically significant impact. In silico A total of 10 PMs (PMI, II, IV–X, and histo-aspartic protease structural and physicochemical inspection of 49c highlighted (HAP)) have been identified in the genome of P. falciparum to poor PK properties emphasizing the need for the development of date. PMs I–IV are transported to the parasitic FV of infected tailored inhibitors with desirable therapeutic properties against erythrocytes, where they orchestrate the degradation of Hb in a Plasmepsin IX/X [26]. 49c was also tested in vivo on other sequential manner with each PM undertaking a distinctive step Plasmodium stages with some efficiency against hepatocyte and in the degradation process, although they are not essential to gametocyte egress as well as interference with the processing parasite survival [15–17]. PMs expressed outside of the FV, PMV– of the cell traversal protein for ookinetes and sporozoites in the X are expressed in all Plasmodium species [17]. PMV, PMIX, and ookinete. Therefore, the absence/inhibition of PMIX and PMX PMX are the only other PMs expressed in the erythrocyte stages. blocks parasite development by impairing egress or invasion PMV is an essential enzyme for survival as it is accountable for of a new erythrocyte. Oral treatment of a novel dual PMIX/X 206 Toxicology Research, 2021, Vol. 10, No. 2 Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021

Figure 1: Malaria infection cycle. The Anopheles mosquito bites a human taking a blood meal and sporozoites from the salivary gland enter the human blood stream. The sporozoites move to the liver and invade hepatocytes. Within the liver they develop to produce exoerythrocytic merozoites which are released into the blood stream. Merozoites invade erythrocytes which takes between 10 and 30 s and involves recognition of the erythrocyte membrane, attachment, reorientation, and entry. Within the erythrocyte merozoites grow into a ring or trophozoite and mature in to a schizont. The schizont produces merozoites which are primed to invade further erythrocytes. The release of merozoites, known as egress, is a two-step process: the degradation of the parasitophorous vacuole, which encloses the merozoites, and the degradation of the erythrocyte membrane. Gametocytes are formed from the asexual blood stage and are taken up by a feeding mosquito. The gametocytes mature in the mosquito gut to become male and female gametes. The fertilized zygote develops to an ookinete and an oocyst inside the mosquito. Sporozoites then migrate to the salivary glands where they can be injected into a new human host to propagate the spread of infection. Adapted from Cowman et al. [21] and Weiss et al. [24]. inhibitor WM382 cured mice of the rodent Plasmodium parasite on different roles in a general acid–base or ‘push and pull’ P. berghei and prevented blood infection from the liver. In addition, reaction [32]. In spite of the heterogeneity among aspartic pro- WM382 was efficacious against P. falciparum asexual infection in teases, the (motif Asp-Ser/Thr-Gly) and mechanism of humanized mice and prevented transmission to mosquitoes action is conserved throughout the family [32–34]. The high levels [10]. No toxicological endpoints were evaluated in any of these of conservation between Plasmodium and mammalian aspartic studies. proteases has directed lead optimization studies to focus on improving PM inhibition selectivity versus the related human Potential toxicities of inhibiting mammalian aspartic proteases [35]. The potential toxicity risks associated with mammalian aspartic proteases aspartic protease inhibition were elucidated through conducting Target-related safety issues are responsible for many drug project a thorough assessment of the literature and publicly available failures [2], therefore, evaluating the potential for on target- data, to produce a target safety assessment (TSA) [33, 36, 37]. related toxicity, in conjunction with developing understanding of Though primarily an in cerebro exercise, the goal of a TSA efficacy, can aide in decision making through the drug discovery is to identify potential unintended consequences of target process. In this case, the proposed targets PMIX/X are specific modulation and to propose a risk evaluation and mitigation to Plasmodium species; nonetheless there is the potential for strategy to assist in early program advancement and to antici- drugs that target PMIX/X to also inhibit human aspartic proteases pate, monitor, and manage potential clinical adverse events (AEs) due to their homology. Mammalian aspartic proteases include [33, 36, 37]. TSAs bring together target homology information, the digestive enzymes (), the intracellular D gene and protein expression profiles, transgenic and mutation (CatD) and (CatE), and . Aspartic proteases are phenotype data, including loss- or gain-of-function within important in hydrolytic processes and have become drug targets mouse/rat and human phenotypes, and understanding of the for several diseases including malaria. For instance the inhibition role of the target under ‘normal’ physiological conditions, as of renin in hypertension treatment [27], human immunodefi- well as in disease state. By combining these data with any public ciency virus (HIV) aspartyl protease inhibitors for autoimmune domain information on competitor compounds, key risks can be deficiency syndrome (AIDS) [28], CatD targeting antibodies in identified and categorized by organ/tissue and/or physiological breast cancer [29], and BACE1 inhibitors for Alzheimer’s disease functions as the basis of a risk mitigation plan with ranking of [30]. Initially, aspartic proteases are synthesized as proenzymes risks and potential next steps [38]. For this specific TSA, data (zymogens) and are activated upon cleavage of the pro-segment were collated on in vitro investigations and in vivo animal studies [31]. Aspartic proteases belong to a class of protease enzymes highlighting mammalian aspartic protease function together which use a catalytic dyad to cleave peptide tetra- with actual adverse drug reactions (ADRs) noted in patients hedral intermediate. The catalytic aspartic acid residues take treated with approved aspartic protease inhibitors (Fig. 2). Barber et al. 207 Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021

Figure 2: Potential risks of mammalian aspartic protease inhibition. An in cerebro and in silico target safety assessment of publicly available literature was performed to uncover the possible toxicity risks to organ systems in the body. As plasmepsins are aspartic proteases specific to Plasmodium, the associated toxicity risks of targeting them would arise from nonspecific host aspartic protease inhibition. A compound with a lack of specificity to plasmepsin IX/X may interact with mammalian aspartic proteases due to their structural similarity, potentially leading to toxicity. Based on the known adverse reactions to existing compounds, along with in vitro and in vivo data, the probability of organ toxicity occurrence was determined. A scale of low (green box), medium (amber boxes), or high (red boxes) was used to categorize the probability of toxicity occurrence. The impact of possible toxicities to progression of the compound if they did indeed occur was assessed based on thetypeandseverity of the effect on target organ inhibition.

Cathepsin D It is a devastating neurodegenerative disorder characterized by severe neurodegeneration, developmental regression, visual loss, CatD is a mammalian aspartic endo-protease that is ubiquitously epilepsy, and premature death [47, 48]. CatD inhibitors have yet distributed in lysosomes [39]. CatD degrades proteins and acti- to reach the clinic; mice used for preclinical xenograft models of vates precursors of bioactive proteins in pre-lysosomal compart- CatD targeting antibodies showed no overt toxicity as indicated ments [40]. The many physiological functions of CatD include by an absence of weight loss although no specific toxicity end- metabolic degradation of intracellular proteins, activation and points were measured [25]. degradation of polypeptide hormones and growth factors, acti- Several structurally distinct beta-secretase (BACE1) inhibitors vation of enzymatic precursors, processing of enzyme activa- have been withdrawn from development after inducing ocular tors and inhibitors, brain antigen processing, and regulation of toxicity following chronic treatment in animal models; CatD apoptosis [41–44]. CatD can also be found in the extracellular was identified as a principal off-target of these BACE1 inhibitors space [45]. CTSD is the gene that encodes CatD; homozygous in human cells. Quantitation of CatD target engagement in deletion of Ctsd in mice leads to progressive atrophy of the cells has been shown to be predictive of ocular toxicity in vivo, intestinal mucosa, increased apoptosis in the thymus and pro- suggesting that off-target inhibition of CatD is a principal driver found destruction of lymphoid cells with early lethality in the of ocular toxicity for BACE1 inhibitors [49]. Therefore, inhibition postnatal phase [46]. These data indicate that CatD is required of CatD could potentially result in a range of toxicological effects in certain epithelial cells for tissue remodelling and renewal. including epithelial cell remodelling and renewal which result Human deficiency of CatD has been reported as an underlying in toxicity to the GI tract, the eye, and congenital abnormalities. cause of congenital human neuronal ceroid-lipofuscinosis (NCL). As CatD has such a broad physiological role in many tissues NCL is a lysosomal storage disease that results from excessive it is difficult to predict which target organ of toxicity would accumulation of lipopigments (lipofuscin) in the body’s tissues. predominate with CatD inhibition. 208 Toxicology Research, 2021, Vol. 10, No. 2

Cathepsin E thereby leading to hypotension, and reduced perfusion pressure in the kidneys which could potentially result in acute kidney CatE is another major mammalian intracellular aspartic injury. proteinase implicated in the physiological and pathological degradation of intracellular and extracellular proteins. It is an intracellular non-lysosomal glycoprotein that is mainly found in Pepsin the skin and in immune cells. It is found at highest abundance Pepsin is the major enzyme present in the mammalian stomach on stomach epithelial mucus-producing cell surfaces [50]. CatE and is the first protease that food proteins encounter in the also plays an important role in immune responses since it is digestive tract. It is involved in protein digestion and its proposed implicated in antigen processing via the major histocompatibil- physiological function is the stimulation of the disintegration of ity complex (MHC) class II pathway [50]. Mice with homozygous the food bolus by fast hydrolysis of intact proteins, rather than knockout of Ctse, the CatE gene, show an increased susceptibility finely digesting the proteins into absorption-ready peptides [57]. to bacterial infection associated with decreased expression of There is no evidence in the literature from transgenic animal

multiple cell surface Toll-like receptors [51]. Deficiency of CatE Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 models or human mutation phenotypes to highlight the effect in mice has also been shown to cause atopic dermatitis (AD), a of pepsin inhibition on normal biology. However, inhibition of pruritic inflammatory skin disease [52]. The reduced expression pepsin could potentially result in a decrease in the stimulation of CatE was also observed in erythrocytes of both humans with of digestion which could lead to a decrease in gastric emptying AD and in an AD mouse model indicating that CatE deficiency and nutrient absorption. might therefore be linked to the induction of AD. CatE deficiency in homozygous knockout mice also induces a form of lysosomal Napsin A storage disorder characterized by accumulation of lysosomal membrane sialoglycoproteins (glycoproteins which contain Napsin A is an aspartic protease present in the epithelial cells sialic acid as one of their carbohydrates), and the elevation of of the mammalian lung and kidney [58]. Expression is seen in lysosomal pH in macrophages. These striking features were also type II alveolar cells of the lung [59], where it is involved in the found in wild-type macrophages treated with pepstatin A (a processing of surfactant protein B (SP-B) [60], and in the renal potent inhibitor of aspartyl proteases), and Ascaris inhibitor proximal tubules where it functions as a lysosomal protease, (a strong inhibitor of CatE but not CatD) [53]. These results highlighting a role in protein catabolism [61]. SP-B plays a critical suggest that CatE is important for preventing the accumulation role in the functioning of healthy lungs; its absence leads to of sialoglycoproteins that can induce a form of lysosomal storage lung conditions, the most common of which is acute respiratory disorder. Therefore, as CatE plays an important role in the main- distress syndrome [62]. Inhibition of Napsin A could potentially tenance of immune system homeostasis by participating in host lead to protein overload in proximal tubules, which is ultimately defence mechanisms, inhibition of CatE could potentially result knowntoleadtotubulointerstitialdamage[63]. Therefore, inhibi- in increased incidence of infection and dermatitis. Furthermore, tion of Napsin A could potentially result in lung or kidney toxicity. CatE inhibition could potentially lead to a lysosomal storage disorder in macrophages. However, as the aim of this project is Beta-secretase enzyme to develop a PMIX/X inhibitor that functions as a single dose There are two forms of the beta-secretase enzyme, BACE1 treatment, the risk of these toxicities is significantly reduced. and BACE2, which were initially identified as transmembrane Renin aspartyl proteases cleaving the amyloid precursor protein (APP) at the β-site [64]. BACE1 is highly expressed throughout the Renin (angiotensinogenase) is a mammalian aspartic protease brain, whereas BACE2 is expressed at low levels in the brain protein and enzyme secreted by the kidneys. It participates but expressed at varying levels in peripheral tissue [31]. BACE1 in the body’s renin–angiotensin–aldosterone system (RAAS) is an aspartic-acid protease important in the formation of that mediates the volume of extracellular fluid, and arterial myelin sheaths in peripheral nerve cells [65]. Due to the cleavage vasoconstriction, thereby regulating the mean arterial blood specificity and localization of BACE1 activity, BACE1 is believed to pressure [31, 54]. Renin is secreted from specialized granular be the secretase responsible for the formation of plaques in the cells found in the juxtaglomerular apparatus of the kidneys, brain. BACE cleaves the APP at the β-secretase site, a critical step and is secreted in response to decreased arterial blood pressure, in the Alzheimer’s disease pathogenesis. Comparison of BACE1 decreased sodium chloride levels, and sympathetic nervous to other aspartic proteases such as CatD and CatE, Napsin A, system activation. Renin is classically termed as a hormone, pepsin, and renin revealed little similarity with respect to the although renin is an enzyme responsible for the hydrolysis of substrate preference and inhibitor profile. On the other hand, the amide bond at L10–V11 of angiotensinogen (from the liver) these parameters are all very similar for the homologous enzyme to angiotensin I which is converted to the potent vasoconstrictor BACE2 [66]. angiotensin II [55, 56]. Angiotensin II leads to the constriction of BACE1- and BACE2-deficient mice demonstrate a wide range blood vessels, increased secretion of antidiuretic hormone and of physiological substrates and functions for both proteases aldosterone; all causes an increase in blood pressure. Renin’s both within and outside of the nervous system. For BACE1 primary function is therefore to cause an increase in blood this includes axon guidance, neurogenesis, muscle spindle pressure, leading to restoration of perfusion pressure in the formation, and neuronal network functions, whereas BACE2 has kidneys. There are several renin inhibitors and angiotensin been shown to be involved in pigmentation and pancreatic β- II receptor antagonists on the market for the treatment of cell function [64]. It is likely that many of the developmental a range of cardiovascular disorders, including hypertension BACE1 functions, such as myelination, would not be affected and heart failure, nephropathy (diabetic and nondiabetic), and in adult patients, with the impact of inhibition occurring in atherosclerosis. They are safe and well tolerated in these patient young children. However, neurogenesis and axon guidance in populations [27]. Inhibition of renin could potentially result the hippocampus would be relevant for all patient populations. in a diminished response to decreased arterial blood pressure Muscle spindle maintenance is an adult BACE1 function and is Barber et al. 209

compromised in adult mice treated with a BACE inhibitor [67]. Therefore, inhibition of BACE1 could compromise brain function and muscle spindle maintenance whereas inhibition of BACE2 could potentially result in hypopigmentation or a perturbed pancreatic β-cell function.

Toxicities associated with approved aspartic protease inhibitors One of the most well-known protease-based therapies is the HIV aspartic protease inhibitors, which are widely used in the combined treatment of AIDS. These inhibitors block the crucial viral maturation stage and thereby reduce the spread of HIV.Such inhibitors approved by the FDA include saquinavir, ritonavir, Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 indinavir, nelfinavir, lopinavir, atazanavir, amprenavir, fosam- prenavir, tipranavir, and darunavir [28]. It has been shown that saquinavir, ritonavir, and indinavir, at therapeutically relevant concentrations, inhibit the growth of P. falciparum in vitro cell cultures [68, 69]andin vivo in mice [70, 71]. AEs in response to treatment with the main subclasses of highly potent antiretroviral therapy have recently been reviewed [72]. Aspartic protease inhibitors resulted in the largest number of reported AEs caused by continuous use. Most AEs were related to hyperlipidaemia but other AEs included renal, gastrointesti- nal, and dermatological toxicities [73, 74]. In one case, aspartic protease inhibitors were linked to the development of anaemia in post-partum women [72]. The main AEs of ritonavir include vomiting, nausea, and abdominal pain at onset of treatment. An increase in the concentration of ritonatir has also been associated with hypertriglyceridaemia [72]. AEs reported after 48 weeks of treatment with tipranavir/ritonavir include gastrointestinal symptoms and grade 3–4 elevations in cholesterol levels [75]. The use of atazanavir/ritonavir also resulted in a less favourable lipid profile with a significant increase in plasma triglyceride levels [76]. Clinical trials with HIV aspartic protease inhibitors show that major AEs can occur during treatment, necessitating interrup- tion and/or changes of medication. The AEs are thought to be due to the inhibition of human aspartic proteases such as CatD.

For example, interactions between ritonavir and nonretroviral Figure 3: Non-clinical safety assessment plan. A non-clinical safety assessment aspartic proteinases are well established, with Ki values against plan was formulated to test the selectivity of compounds progressing in the pro- human aspartic proteases CatD and CatE of 20 and 8 nmol/l, gramme for plasmepsin IX/X versus mammalian aspartic proteases. First in the respectively. The plasmepsins are much more closely related selectivity testing cascade, a high-throughput assay to understand direct inhibi- to human CatD than to HIV proteinase so if development of tion of mammalian aspartic proteases in comparison to plasmepsins should be performed. Then a cell-based assay would provide a more accurate reflection of antimalarial therapy based on HIV proteinase inhibitors is con- compound potency. Combining in vitro IC50 values with pharmacokinetic data to sidered, the potential interactions with the human aspartic pro- model target engagement would further develop understanding of the specificity teinases may need to be minimized [77]. profile. A comparison of lead compounds with compounds known to interact with mammalian aspartic proteases may also be useful in guiding the chemistry. An investigative in vivo toxicity study, in combination with an understanding of Nonclinical safety assessment plan exposures required for efficacy, would be useful to identify and understand the selectivity window against mammalian targets required. Endpoints for the in vivo A nonclinical safety assessment plan was formulated to test toxicity study would be chosen based on the findings from the TSA and in vitro the selectivity versus the intended therapeutic target of com- studies. Before the first-in-human trial, an assessment in both a rodent and non- pounds progressing in the programme (Fig. 3). The chemistry of rodent species for a duration sufficient to support the initial clinical protocol will the compound should be driven by prioritizing selectivity against need to be conducted. A combination of a range of in vitro and in vivo studies CatD and CatE inhibition since these are likely to have the most is crucial to understand the overall safety profile and ensure progression of the compound(s) in the programme. undesirable and severe effects. Initially, an isolated enzyme assay could be used as a high- throughput screen to assess direct inhibition of mammalian of the selectivity profile. However, when designing the com- aspartyl proteases, including CatD and CatE. Translating these pounds, understanding the selectivity window in these assays inhibition values to the context of plasmepsin potency can be sufficiently to drive it wider with each chemistry optimization challenging, due to technical differences between assay set- round is a pragmatic approach. It is not possible to predict up such as platforms used, concentrations of protein and sub- adverse outcomes from the isolated biochemical enzyme in vitro strates. Thus, a direct comparison may not be a true reflection IC50 value alone, as the duration of inhibition (both in terms 210 Toxicology Research, 2021, Vol. 10, No. 2

of hours in the day and number of days) will determine the administration in women of child-bearing potential and children, incidence, severity, and reversibility of toxicity. From the in vivo key patient populations for an antimalarial therapy. pharmacokinetic (PK) data, it may be possible to predict levels of off-target engagement, and also get some understanding of likely Discussion toxicities, as described by Zuhl et al.[49]. A lower throughput, more quantitative method may have the Malaria remains a highly challenging problem with >200 million potential to provide a more accurate determination of inhibitor cases annually. Since the malaria parasite routinely develops potency and could be used to assay newly identified targets in resistance to drugs, there is a continual need for novel antimalar- live cells [49]. In their assay Zuhl et al., reported that several ials to combat the disease. Development of a small molecule BACE1 inhibitors blocked activity of CatD in a cell-based assay to plasmepsin IX/X inhibitor presents a unique opportunity to a much greater potency compared to the isolated enzyme assay, specifically target the malarial aspartic proteases. Furthermore, indicating that results in the more sophisticated assay are depen- focusing on parasite-specific targets for which no human dent on more than just the kinetics of enzyme inhibition. This host orthologue exists reduces the chance of drug toxicity. can also happen in the reverse, with cellular potency dropping However, since this is a small molecule programme rather than Downloaded from https://academic.oup.com/toxres/article/10/2/203/6135370 by guest on 30 September 2021 off compared to isolated enzyme potency, due to factors such a vaccine or antibody approach, there may be lack of specificity, as cellular efflux and/or permeability. Another approach is to introducing the potential for risks associated with hitting related design a reporter substrate that is primarily cleaved by the target mammalian targets. protease in a complex cellular extract or even in intact cells [78]. We identified a range of mammalian proteins that inhibitors Again, however, it is not possible to predict adverse outcomes of plasmepsin IX/X may interact with including pepsin, cathep- from the cell based in vitro IC50 value alone, as it does not take sins, β secretases, and renin. This reinforced the need for speci- into account the ADME (absorption, distribution, metabolism, or ficity and provided the necessary data to direct the chemistry excretion) properties of a compound, all of which can influence early in the programme. By assessing gene and protein expres- the toxicological profile. Combining in vitro IC50 values with PK sion, transgenic mouse data, human mutation phenotypes, and data and modelling possible target engagement and outcomes ADRs, we have been able to identify the key risks associated with in comparison to compounds known to cause the toxicity by this the project. The risk evaluation and mitigation strategy suggest mechanism may be useful. that a thorough evaluation of the selectivity profile coupled with Cell-free and in vitro assays are useful to understand selectiv- an early in vivo evaluation will be vital to anticipate, monitor, ity and predict what may happen in vivo. But ultimately, an in vivo and manage potential clinical AEs. Additionally, since the project assessment in the rodent early in the programme, when there are aim is to develop a single-dose treatment, such a study will be tool compounds with suitable in vivo PK properties, would help important to help define onset, dose-dependency, reversibility, identify and understand the selectivity window against mam- and margin of safety. malian targets that may be required in context with exposures required for efficacy. Both rat and mouse aspartic proteases have Conclusion a high level of homology with the human orthologs on a gene and protein level (data not shown). Mammalian aspartic pro- Compared with TSAs conducted for drug projects in many other teases also share topologically similar domains and active site therapy areas, TSAs for malaria differ since there is often no residues [33]. Together this suggests that the rodent enzymes are identical mammalian target. However, the overall conclusion sufficiently similar in catalytic site structure and function to be remains the same as for other therapy areas; use all available able to make useful selectivity comparisons for small molecule data to generate an early risk management plan that can identify compounds. Endpoints to consider (depending on the enzyme and mitigate any potential toxicological issues in the project. selectivity profile if known) include cholesterol and triglyceride levels, visual acuity assessment and assessment of accumulation of autofluorescent material in the retinal pigmented epithelium Conflict of interest statement (RPE), histopathology of major organs (stomach, intestine, thy- RR is a director and cofounder of ApconiX, an integrated toxi- mus, skin, kidney, lungs, liver), CNS functional endpoints, blood cology and ion channel company that provides expert advice on pressure assessment, and potentially assessment of aspartic non-clinical aspects of drug discovery and drug development to protease levels in tissues with lesions. Based on the predicted academia, industry, and not-for-profit organizations. RR, JB, CS toxicities, special stains such as Schmarl’s stain for lipofuscin and PS are employees of ApconiX. Clients of ApconiX include UCB may be required. Even with a well-designed experiment it could Biopharma SRL and MMV. JPV is an employee and stockholder of be difficult to capture all these endpoints in a single experiment, UCB Biopharma SRL. therefore, depending on the selectivity profile of the compounds being tested, prioritization of endpoints may be required. Depending on the overall selectivity profiles of the com- References pounds, the results from an initial in vivo investigative assess- 1. Wong CH, Siah KW, Lo AW. Estimation of clinical trial success ment, the clinical plan, the planned patient population, and the rates and related parameters. Biostatistics 2019;20:273–86. overall confidence in and appetite for risk within the programme, doi: 10.1093/biostatistics/kxx069. decisions on the progression of a compound(s) for further eval- 2. Cook D, Brown D, Alexander R et al. Lessons learned uation can be made. 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