Repurposing elesclomol, an investigational drug for the treatment of copper metabolism disorders

1. Introduction

Copper serves as a structural and catalytic cofactor for a variety of enzymes in aerobic organisms. Though present in trace amounts, copper is essential for numerous biochemical processes including mitochondrial energy generation, free radical eradication, neurotransmitter synthesis, neuropeptide maturation, connective tissue formation, iron homeostasis, and pigment production [1]. Owing to its diverse roles in cellular and organismal physiology, it is not surprising that copper deficiency can result in multi-systemic diseases. Indeed, loss-of-function mutations in copper transport proteins result in debilitating and frequently fatal infantile disorders [2]. Amongst the many rare genetic disorders of copper deficiency, the most prevalent and best studied is Menkes disease, which is an X-linked disease caused by mutations in the copper transporting ATPase, ATP7A [3]. Menkes is a relatively rare disease with incidence estimates ranging from 1/50,000 to 1/360,000 live births depending on the population surveyed [3]. Loss of functional ATP7A, which is required for directional transport across polarized epithelial cells such as the intestinal enterocytes, results in systemic copper deficiency due to impaired absorption of dietary copper. Copper deficiency in Menkes children manifests in characteristic kinky hair, hypopigmentation, connective tissue abnormalities, seizures, and progressive neurodegeneration, with death typically occurring by the third year of life [3]. Partial loss-of-function ATP7A variants have been identified in patients with occipital horn syndrome (OHS), which is a milder form of Menkes disease [4]. Like Menkes patients, OHS patients display hair and connective tissue abnormalities, but their neurological symptoms are milder [4]. Additionally, missense mutations in the carboxyl half of ATP7A have been associated with X-linked distal hereditary motor neuropathy, and mutations in AP1S1 (σ1A subunit of the adaptor protein complex I), which disrupt intracellular trafficking of copper transporters ATP7A and ATP7B, have been found in patients with MEDNIK syndrome [2]. Currently, no effective therapy exists for these disorders, though early administration of copper-histidine could be beneficial to a subset of Menkes patients, especially, if the mutant ATP7A retains some residual activity [5].

2. Rationale

Treating Menkes patients by parenteral copper supplementation using hydrophilic complexes, such as copper histidinate, fails to ameliorate disease pathology in most cases [5]. This is because hydrophilic copper salts are unable to reach cuproenzymes present in different subcellular compartments of various tissues, especially the brain. Administration of supra-physiological levels of copper is toxic to cells, as copper has a strong tendency to generate reactive oxygen species (ROS) [6]. To counteract copper toxicity, organisms have evolved a highly regulated network of copper-transporting, copper-escorting, and copper-storing proteins that tightly control copper levels in cells [1]. Thus, site-specific delivery of copper to cuproenzymes has to overcome two major obstacles: First, it must cross multiple biological membranes, and, second, it must bypass the regulatory network of copper-trafficking/storing proteins. In this regard, a membrane-traversing drug capable of binding copper outside the cell and releasing it inside the cell might prove more effective in delivering copper to intracellular cuproenzymes. This “Trojan-horse” fashion delivery of metals by other lipophilic carrier molecules, such as the iron-transporting hinokitiol, has shown promise in restoring iron levels in preclinical animal models [7]. Using this principle, Soma et al., performed a targeted screen with a number of copper-binding pharmacological agents for their ability to restore respiratory growth in a yeast mutant defective in copper delivery to cytochrome c oxidase (CcO), a mitochondrial cuproenzyme [8]. This screen identified elesclomol as the most potent copper-transporting molecule, which rescued CcO function in a number of in vitro and in vivo models of copper deficiency [8,9].

3. Elesclomol as a copper-transporting therapeutic agent

Elesclomol is a bis(thiohydrazide) amide compound that binds copper in a 1:1 ratio (Fig. 1). It was originally developed as a chemotherapeutic agent by Synta Pharmaceuticals. The parent compound of elesclomol was identified through a cell-based multidrug resistance modulators screen and subsequent structure-activity relationship studies led to the synthesis of elesclomol [10]. Preclinical studies showed that the intrinsic antitumor activity of elesclomol is low, but it significantly enhances the antitumor activity of paclitaxel [10]. Therefore, elesclomol has been evaluated in a number of clinical trials in a combination treatment with paclitaxel for anticancer activity [11–13]. Mechanistically, elesclomol binds copper(II) in the extracellular environment and forms a membrane permeable complex, which upon entering the mitochondria releases copper after it is reduced to copper(I), a reaction likely mediated by mitochondrial ferredoxin 1 [14,15] (Fig. 1). Copper(I) released in the mitochondria can react with molecular oxygen generating ROS, which when produced in excess, can cause unmitigated oxidative stress and apoptotic death of cancer cells [14]. While this mechanism of action is consistent with its anticancer activity, it was recently shown that copper(I) released from elesclomol also becomes available for the metalation of CcO [8]. Consistently, low doses of elesclomol were shown to safely but effectively deliver copper to CcO and restore mitochondrial function in genetic models of copper deficiency [8].

Figure 1. Elesclomol: a bis(thiohydrazide) amide compound that binds copper in a 1:1 ratio.

Elesclomol (ES) binds copper (Cu²⁺) in the extracellular environment and transports it to the mitochondrial matrix. ES-Cu(II) complex is then reduced to ES and Cu(I) in the mitochondrial matrix in a reaction likely catalyzed by ferredoxin 1 (FDX1). The copper(I) thus released can generate reactive oxygen species (ROS). This copper(I) has been shown to be bioavailable for the metalation of cytochrome c oxidase (CcO) (solid arrow) and is also likely to be delivered to other subcellular compartments (dotted arrows), where it can be used for the maturation of cuproenzymes, including superoxide dismutase 1 (SOD1) in the mitochondria and cytosol and lysyl oxidase (LOX), dopamine β-hydroxylase (DBH), ceruloplasmin, peptidylglycine α-amidating mono oxygenase (PAM), and tyrosinase in the secretory pathway.

4. Expert Opinion

Organisms have evolved exquisite systemic and cellular mechanisms to maintain homeostasis of highly reactive metals like copper. Genetic mutations in uptake, distribution, and excretory machinery that perturb this homeostasis in either direction can result in disease states. For example, profound copper deficiency is seen in the case of Menkes disease, whereas excess copper accumulation results in Wilson’s disease [16]. Thus, pharmacological interventions that can restore cellular and tissue metal homeostasis could be of therapeutic benefit [17]. Indeed, such approaches have often been used for metal chelation therapy [18], and more recent efforts have focused on restoring the site- and direction-specific transport of metals, as in the case of hinokitiol for iron and elesclomol for copper homeostasis [7–9].

A recent preclinical study showing the efficacy of the elesclomol-copper complex in alleviating neuropathology and mortality in a mouse model of Menkes disease is an exciting development [9]. This is because: 1) the mottled-brindled (mo-br) mouse used in this study recapitulates severe pathology observed in Menkes-affected children, 2) like Menkes patients, mo-br mice are particularly recalcitrant to treatment with hydrophilic copper complexes such as copper-histidinate, 3) just two injections of elesclomol-copper complex extended the median survival of mo-br mice from 14 to 203 days, and 4) along with extended survival, the treated mice thrived as indicated by the normalization of many aspects of animal physiology, biochemistry, and anatomy, including neuromotor function, CcO levels, and hair texture.

While this promising preclinical study [9] opens up a new avenue for the treatment of this devastating disorder, it is important to identify the next steps in repurposing elesclomol for the treatment of copper deficiency disorders. First, it is critical to determine the exact dosing regimen such that the toxicity of this anticancer drug is minimized while optimal efficacy is achieved. The expected cause of toxicity is delivery of excess copper to mitochondria and subsequent elevation in ROS-mediated cell death. This could be mitigated by the co-administration of antioxidants. Indeed, at the cellular level it has been shown that increased ROS generated by elesclomol can be neutralized by pre-treatment with common antioxidants, including N-acetyl cysteine [19]. Interestingly, unlike cancer cells, elesclomol toxicity is minimal in healthy cells, suggesting that the endogenous antioxidant capacity of healthy cells may overcome potential toxicity issues [8,14]. Consistently, clinical trials with cancer patients administered high doses of elesclomol have shown that it is well tolerated [11–13]. However, it should be noted that in these studies, elesclomol was not used in combination with copper, which will be required when repurposing it for copper-deficiency diseases like Menkes. Therefore, careful toxicity studies with the elesclomol-copper complex will be needed. Along with monitoring toxicity, biomarkers of efficacy will have to be defined. One of the prominent neurological presentations in Menkes infants is intermittent seizures [3], which could serve as an important biomarker of clinical efficacy. In terms of blood-based biomarkers, the levels of catecholamines such as dopamine and norepinephrine in plasma could be useful. This is because the activity of the copper-dependent enzyme dopamine β-hydroxylase, which is required for the conversion of dopamine to norepinephrine, is reduced in Menkes infants [5]. A previous study has shown that the ratio of dopamine to norepinephrine and the ratio of their breakdown products dihydroxyphenylacetic acid to dihydroxyphenylglycol in patient plasma are reliable diagnostic markers for the disease [5]. Correction of dopamine β-hydroxylase activity with drug treatment is expected to normalize these neurochemicals, making them valuable biomarkers of efficacy.

The second critical question is the timing and frequency of treatment. The mo-br mice study showed that two injections of elesclomol-copper early in life were sufficient to prolong the lifespan of these Menkes-affected mice [9]. In terms of translating this observation in human patients, it will be important to determine how early in life the treatment has to be initiated. Studies from copper-histidinate treatment suggest that there is a therapeutic window under which treatment is most likely to be effective [5]. This window is within a few months after birth. Therefore, early diagnosis of the disease would be critical. On that front, targeted next-generation sequencing of the newborns could be used to accurately detect the disease [20]. In terms of determining the frequency of treatment, biomarkers of copper levels in the blood, such as ceruloplasmin and catecholamines, could be useful.

Third, determining the efficiency of elesclomol in delivering copper to cuproenzymes residing in different subcellular compartments will be important for its application to different copper deficiency disorders. Studies showing the rescue of mitochondrial CcO in copper-deficient cells in culture and mice are very promising [8,9], but it would also be important to determine whether elesclomol can deliver copper to cuproenzymes in other subcellular compartments, especially the Golgi body, where a number of cuproenzymes are metalated (Fig. 1). The partial rescue of skin pigmentation and normalization of whisker morphology in elesclomol-copper treated mo-br mice suggests that copper is also delivered to the Golgi compartment, as the copper-dependent enzymes tyrosinase, which is required for melanin production, and sulfhydryl oxidase, which is required for normal hair morphology, receive their copper in the Golgi body. How elesclomol impacts the partitioning of copper in cells and tissues will be particularly important for its application to other disorders of copper metabolism where copper is directly or indirectly implicated in pathogenesis, including OHS, X-linked distal hereditary motor neuropathy, MEDNIK syndrome, Wilson’s disease, aceruloplasminemia, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and a subset of mitochondrial disorders caused by mutations in genes involved in copper delivery to CcO [2, 21, 22]. Notably, the efficacy of Cu(II)ATSM, another copper ionophore, in the treatment of ALS is being evaluated in an ongoing clinical trial, NCT04082832.

An experimental therapeutic strategy involving a combination of cerebrospinal fluid-directed viral gene therapy with subcutaneous administration of copper histidinate has shown promise in a preclinical mo-br mouse model of Menkes disease [23]. However, unlike small molecules like elesclomol, which can correct copper deficiency throughout the body, the proposed gene therapy approach is limited to the brain, leaving other tissues uncorrected. While there is significant therapeutic potential, gene therapy approaches still present several challenges with regards to gene delivery, safety, efficacy, and costs before they become more common in clinical use [24].

Thus, there is an urgent need for the development of an effective therapy for copper deficiency diseases like Menkes, for which currently no Food and Drug Administration-approved drug is available. Direct administration of copper-histidinate alone has not proved to be effective; therefore, more selective copper delivery via the hydrophobic membrane permeable elesclomol-copper complex represents a promising approach, especially considering that elesclomol has already undergone clinical trials and information about its pharmacokinetics, toxicity, and drug metabolism is available.