Steroid sulfatase inhibitors and sulfated C19 steroids for proteotoxicity-related diseases: a patent spotlight

Abstract

Aging and proteotoxicity go hand in hand. Inhibiting proteotoxicity has been proposed to extend lifespan. This invention describes a new strategy to limit proteotoxicity and to extend the lifespan. Loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase, elevates the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. The present invention provides a group of molecules for use in the prevention of aging-associated proteotoxicity caused by protein aggregation diseases and/or to increase the lifespan of a eukaryotic organism. These molecules are either steroid sulfatase inhibitors or sulfated C19 steroids, both of which reproduce the phenotype of sul-2 mutants. One particular representative example is STX-64. Potential applications of the claims have been demonstrated in animal models of Parkinson's disease, Huntington's disease and Alzheimer's disease.

Background

Proteotoxicity refers to the dysfunction of cells caused by the misfolding or aggregation of proteins. It is a significant characteristic observed in various neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, frontotemporal dementia, amyotrophic lateral sclerosis and Huntington's disease, among others. These diseases often involve neuronal damage resulting from abnormal protein aggregation and deposition. The aging process is a key risk factor for neurodegenerative diseases, and during aging, proteotoxicity becomes more prevalent due to a decline in our ability to maintain protein balance. Numerous studies have demonstrated that aging actively hinders our capacity to clear harmful protein aggregates, leading to their accumulation and initiating neurodegeneration later in life [1]. Ultimately, proteotoxicity imposes constraints on the lifespan of both humans and animals. This invention presents a novel approach to address proteotoxicity, with the potential to extend lifespan.

Activating various genetic pathways can lead to an extension of animals' lifespans. This process is carefully regulated and involves the coordination of tissues and environmental signals. Hormones play a crucial role in facilitating communication between cells and tissues, making them significant contributors to the regulation of longevity. Among the hormones involved in different pathways that impact longevity are insulin and IGF, TGF-β and dafachronic acids (DAs). Studies have shown that these hormones influence the lifespan of the model organism Caenorhabditis elegans [2]. Along these lines, the gonad serves as an endocrine tissue that produces steroid hormones to regulate various physiological aspects, including longevity. In C. elegans, germline ablation has been found to extend the organism's lifespan. Several factors contribute to this longevity-promoting effect, including the synthesis of DA by the somatic gonad, the involvement of the transcription factor encoded by daf-16 (a homolog to the human FOXO gene), and the nuclear receptors encoded by genes daf-12, nhr-80 and nhr-49 [3].

Steroid hormones are not limited to production solely in gonads; they can also be synthesized in other tissues. Within the central nervous system (CNS), these steroid hormones are referred to as neurosteroids. Neurosteroids play a significant role in modulating neurotransmission both directly, by targeting neurotransmitter receptors, and indirectly through other mechanisms [4]. One noteworthy aspect is that steroid hormones can undergo sulfation by a sulfotransferase enzyme, resulting in a significant alteration of their chemical properties. This modification impairs their function as hormone receptor activators. Interestingly, sulfated steroid hormones can still act as neurosteroids, effectively regulating neurotransmission [4]. Among the sulfated steroid hormones, dehydroepiandrosterone sulfate (DHEAS) has long been associated with aging. As individuals age or experience age-related conditions such as sarcopenia or Alzheimer's disease, the levels of DHEAS tend to decline. This observation has led to speculation regarding its potential causative role in these age-related conditions [5].

A group of patents has put forth a claim suggesting that inhibiting steroid sulfatase can lead to an increase in the levels of sulfated hormones, resulting in extended longevity and relief from symptoms associated with protein aggregation diseases. The prolonged lifespan seems to be influenced by the same factors previously linked to longevity due to germline ablation. When treated with a particular steroid sulfatase inhibitor, the beneficial effects on longevity and protein aggregation diseases observed in the sul-2 mutant of C. elegans are replicated.

Interestingly, this specific steroid sulfatase inhibitor treatment also improves cognitive symptoms and reduces plaque formation in a mammalian model of Alzheimer's disease. It is noteworthy that using sulfated C19 androgen steroid hormones produces similar outcomes as those induced by steroid sulfatase inhibition. However, neither the non-sulfated forms nor the sulfated C21 pregnenolone hormone yield the same positive effects. Overall, these patents propose that the use of steroid sulfatase inhibitors or specific sulfated C19 steroid hormones could potentially serve as treatments for aging and aging-related diseases. Such interventions might extend lifespan and provide protection against age-related proteotoxicity [6,7].

Description of the invention & its potential

The invention by the Universidad Pablo de Olavide describes sulfated C19 steroid hormones to treat and/or prevent proteotoxicity in protein-aggregation diseases, extend lifespan and protect against aging-associated proteotoxicity. Experimental findings have demonstrated that inhibiting the activity of steroid sulfatase, either through genetic mutation or by utilizing a specific steroid sulfatase inhibitor, extends the lifespan of C. elegans and provides protection against aging-related proteotoxicity in nematodes [6,7]. During the study, researchers isolated thermotolerant mutants of C. elegans and identified an allele called pv17 of the sul-2 gene, which is one of the three members of the C. elegans sulfatase family. The pv17 allele introduces a single amino acid substitution leading to reduced function. Remarkably, both worms carrying the isolated pv17 allele and those with the null allele gk187 of sul-2 were found to have a longer lifespan compared with the wild-type. Throughout their aging process, pumping frequency and mobility declined at a similar or slower rate than that observed in the wild-type, indicating a healthier lifespan. Interestingly, the deletion of the other two sulfatase genes, sul-1 and sul-3, did not result in increased lifespan, suggesting that the sulfatase sul-2 plays a primary role in regulating longevity.

Clearly, the activity of sul-2 seems to be associated with the modification of sulfated steroid hormones and steroid hormone sulfatases. Researchers have successfully generated specific inhibitor for these types of enzymes, known as STX64 (an orally active nonsteroidal inhibitor of steroid sulfatase that was under development as treatment for hormone-sensitive cancers such as breast cancer, prostate cancer and endometrial cancer) [8]. Notably, the treatment with STX64 has been shown to increase the lifespan of wild-type animals. Moreover, STX64 treatment exhibits similar phenotypes to other sul-2 mutant phenotypes. Interestingly, STX64 did not further enhance the lifespan of sul-2 deletion mutants, suggesting that the increased longevity achieved by STX64 is achieved through inhibiting the sulfatase activity of SUL-2. These observations also imply that SUL-2 can function as a steroid hormone sulfatase, influencing longevity by altering the sulfated state of one or multiple steroid hormones. To investigate whether SUL-2 operates within a known longevity pathway, researchers conducted genetic interaction studies with alleles known to affect longevity. Mutations in the insulin/insulin-like growth factor receptor daf-2 have been previously shown to increase lifespan through the involvement of the transcription factor DAF-16/FOXO12. Interestingly, sul-2 mutations further extended the lifespan of daf-2 reduction-of-function mutants, indicating that sul-2 acts through a distinct pathway to regulate longevity [6,7].

Longevity resulting from the absence of germline requires the involvement of DAF-1 [9], which mainly translocates to the nuclei of intestinal cells. Conversely, in mutants with insulin signaling defects, DAF-16 localizes to the nucleus in most cells [10]. Interestingly, in sul-2 mutants, DAF-16 predominantly localizes to intestinal nuclei, indicating a potential role for sul-2 in germline-mediated longevity. The enhanced longevity observed in sul-2 mutants also depends on other critical factors involved in germline-mediated longevity. Although nuclear hormone receptor NHR-80 has a mild effect on longevity, NHR-49, which is also essential for germline-mediated longevity, shows no impact in sul-2 mutants. Additionally, the deletion of sul-2 has minimal effect on the longevity of the germline-less mutants [11]. According to the claims, these findings collectively suggest that sul-2 acts as a mediator of signaling from the gonad to regulate longevity. Notably, sul-2 mutations do not affect fertility, reproductive age, or gonad morphology, indicating that sul-2 affects a signaling pathway that adjusts lifespan to the reproductive status without influencing gonadal function. Similar to germline ablation, sul-2 mutants further increase longevity in response to dietary restriction, implying that sul-2 is not involved in the longevity induced by dietary restrictions.

Furthermore, the activation of the nuclear receptor DAF-12 by bile acid-like steroids has been shown to trigger an increase in longevity in germline-less animals [12]. Interestingly, the requirement of daf-12 for the extended longevity observed in sul-2 mutants suggests that SUL-2 inactivation leads to changes in the pool of sulfated steroid hormones, generating a signal upstream of DAF-12 that mimics the longevity signals observed in animals with depleted gonads. Another crucial component involved in this process is DAF-36, which converts cholesterol to 7-dehydrocholesterol in the biosynthetic pathway of Δ⁷-DAs [13]. Thus, DAF-36 is also essential for the increased longevity observed in germline-less animals [14]. Likewise, DAF-36 is required for the longevity conferred by the steroid sulfatase inhibitor STX64, indicating that the signal generated by sulfated steroid hormones acts upstream of, or in parallel with, the biosynthesis of Δ⁷-DAs.

Defects in odor sensing as contributing factors to longevity [15] were excluded because the ability of sul-2 mutants to respond to odor was similar to that of wild-type animals. Furthermore, sul-2 mutation increases the longevity of daf-10(m79), a long-lived mutant defective in sensory cilia formation. These results show that the longevity phenotype observed in sul-2 mutants is not due to the impaired functionality of sensory neurons.

The effects of sul-2 mutations and STX64 treatment on C. elegans models for neurodegenerative diseases were investigated. In a C. elegans model of Parkinson's disease, where human α-synuclein expressed in muscle cells causes age-dependent paralysis [16], both sul-2 mutations and treatment with STX64 significantly improved mobility. The loss of SUL-2 function led to a decrease in the number of α-synuclein aggregates, indicating that worms with reduced steroid sulfatase activity handle protein aggregates more effectively. To further assess the neuroprotective effect of reduced SUL-2 activity, a strain expressing α-synuclein in dopaminergic neurons was examined. In this model, labeled dopaminergic neurons died due to α-synuclein toxicity [17]. Remarkably, sul-2 mutants exhibited increased neuron survival compared with control worms, pointing to a neuroprotective action of reduced steroid hormone sulfatase activity. In a neurodegenerative model of Huntington's disease, wherein polyglutamine repeats are fused to a construct that aggregates in adult worms, it was observed that both sul-2 mutation and treatment with STX64 resulted in a reduction in the number of aggregates [18]. Similarly, in two different worm models of Alzheimer's disease, where immobility is induced by the expression of βA protein in muscle cells [19], sul-2 mutation and STX64 treatment consistently delayed paralysis. These findings suggest that inhibiting SUL-2 provides protection to the nematode against aging-related proteotoxicity.

As STX64 ameliorated neurodegeneration in C. elegans models, the effect of this drug on cognitive alterations provoked by intrahippocampal βA oligomers infusion, an acute Alzheimer's disease mammalian model, was also tested. Local and systemic STX64 treatments were found to revert the cognitive deficiencies, measured by passive avoidance test, caused by intrahippocampal administration of βA oligomers [6,7]. Along these lines, it was previously shown that local administration of DU-14, an inhibitor of steroid hormones sulfatase, could also alleviate memory loss caused by intrahippocampal administration of βA oligomers in a mammalian model [20]. Moreover, a study evaluated the effect of 3–4 weeks of oral treatment with STX64 on amyloid deposition in the neocortex (cerebral cortex and hippocampus) of APP-PS1 mice over 15 months old. The goal was to determine the impact of STX64 oral treatment on amyloid pathology in a chronic Alzheimer's disease mice model. The analysis of β-amyloid (βA) plaque density and size showed a significant reduction in mice treated with STX64, except for plaque size in the hippocampus. Additionally, the βA immunoreactive area was reduced in both tissues. Interestingly, STX64 was found to be particularly effective in reducing βA deposition in APP-PS1 mice older than 15 months, compared with APP-PS1 mice aged 10–12 months. Furthermore, the cognitive behavior of the mice was assessed using the passive avoidance test. Vehicle-treated APP-PS1 mice exhibited cognitive deficits, whereas those treated with STX64 completely reversed the cognitive deficiencies, reaching similar levels to those observed in mice under 15 months old. These results collectively indicate that STX64 treatment in APP-PS1 mice reduces βA deposition and leads to improvements in cognitive behavior, even in acute and chronic Alzheimer's disease models. This suggests that STX64 and similar molecules, hold potential as a pharmacological therapy for neurodegenerative diseases.

In order to sort out whether the beneficial effect of SUL-2 inhibition is due to the reduction of non-sulfated hormones or the increase of sulfated hormones, commercially available sulfated steroid hormones were evaluated. It was observed that the C19 androgens DHEAS, testosterone sulfate (TS), and epitestosterone sulfate (ES) improved the mobility in the Parkinson model of C. elegans, with a better result for ES. Similar results were obtained in the Alzheimer's model with TS and ES. Non-sulfated DHEA or non-sulfated testosterone showed no effect, neither pregnenolone sulfate, which belongs to the C21 group of steroid hormones. These results indicated that at least some sulfated C19 androgens are involved in the protective effect against protein aggregation diseases and strongly suggested that the beneficial effect of SUL-2 inhibition is due to the increased levels of this type of hormones. In agreement with these results, treatment with the antiandrogenic compound abiraterone did not affect the wild-type animals but suppressed the beneficial effect of sul-2 mutation. Interestingly, treatment with ES, but not TS or DHEAS, increased longevity on a wild-type background and did not further increase longevity in sul-2 mutants backgrounds, indicating that both interventions share the same molecular mechanism. The addition of ES recapitulated all the phenotypes described for SUL-2 inhibition, strongly suggesting that the causative effect of sul-2 mutation is due to the increase of C19 androgen sulfated hormones related to ES.

The sulfation-mediated regulation of steroid hormones is a conserved process observed in various organisms. In mammals, both sulfotransferases and sulfatases are expressed in different tissues, including the CNS, similar to what is seen in C. elegans. Notably, treatment with a specific steroid sulfatase inhibitor (STX-64), a specific sulfated C19 androgen (epitestosterone sulfate, ES), or a combination of both, tends to improve motor ability, habituation ability, and immediate memory in aged animals. Additionally, short- and long-term memory sessions also showed improvements with the use of the steroid sulfatase inhibitor and/or the sulfated C19 androgen. It is important to mention that vehicle-treated aged mice displayed impairments in learning ability and memory. However, in both memory tests, mice treated with the steroid sulfatase inhibitor, the sulfated C19 androgen, and/or a combination of both exhibited significant improvement in both learning and their ability to store information. Overall, the administration of a steroid sulfatase inhibitor (STX-64), sulfated C19 androgen (ES), or a combination of both significantly improves age-related cognitive impairment, age-associated memory impairment or age-related cognitive decline in animals.

Therefore, an aspect of the present invention refers to a composition, preferably a pharmaceutical composition, nutraceutical composition or dietary supplement comprising sulfated C19 androgens, or any salts or esters thereof, and/or compounds capable of inhibiting the steroid sulfatase of Formula (I) (Figure 1) for use in a method for improving age-related cognitive function and/or treating disorders involving age-related cognitive dysfunction, or age-related cognitive impairment in a subject in need thereof.

Figure 1. . The general structure of steroid sulfatase inhibitors claimed in the patents.

In this structure: R₁-R₆ are independently selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine or astatine), hydroxyl, sulfamate, alkyl and salts thereof. At least one of R₁-R₆ is a sulfamate group, and two or more of R₁-R₆ are linked together to form an additional cyclic structure.

Another aspect of the present invention refers to a method for increasing the lifespan of eukaryotic organisms which consists of: a) providing a sulfated C19 androgen selected from the list of: DHEAS, TS, ES, or androsterone sulfate (AS) (Figure 2A) or b) providing a molecule capable of inhibiting the steroid sulfatase selected from the list consisting of 5-androstene-3β,17β-diol-3 sulfate, DU-14, E1-MTP, EMATE, COUMATE, STX64, KW-2581, STX213 (Figure 2B), RNA-silencing molecules and specific antibody against steroid sulfatase.

Figure 2. . The chemical structures of (A) sulfated C19 steroid and (B) steroid sulfatase inhibitors.

They are claimed to treat and/or prevent aging-associated proteotoxicity caused by protein-aggregation diseases and/or to increase the lifespan of a eukaryotic organism.

Conclusion

Aging and proteotoxicity go hand in hand. Inhibiting proteotoxicity has been proposed to extend lifespan. Sulfated C19 steroid hormones and/or steroid sulfatase inhibitors [6,7,21] are claimed to treat and/or prevent aging-associated proteotoxicity caused by protein-aggregation diseases and/or to increase the lifespan of a eukaryotic organism. Several examples of the two classes of molecules are provided in the patents. The concept was supported by results from animal models of Parkinson's disease, Huntington's disease and Alzheimer's disease and to be established in humans via double-blinded randomized clinical trials.

Future perspective

Neurodegenerative diseases present a great societal burden. These diseases often involve neuronal damage resulting from aging-related proteotoxicity. Steroid sulfatase inhibitors have demonstrated promising results in treating various diseases [22] including neurodegenerative disorders. Furthermore, sulfated C19 steroid hormones also carry a similar potential. To help bring these molecules to clinical use, potential issues such as oral bioavailability, chemical stability and estrogenicity should be overcome. Importantly, the efficacy and safety of the proposed molecules for treating proteotoxicity-related neurodegenerative diseases and for extending the lifespan of humans and animals remain to be elucidated in clinical trials.

Executive summary.

Background

• Aging is associated with proteotoxicity, which limits the lifespan of humans and animals.

• Steroid sulfatase inhibitors increase the percentage of sulfated hormones, increase longevity and improve symptoms related to protein aggregation diseases.

Description of the invention & its potential

• Inhibitors of steroid sulfatase can be of interest for treating aging and aging-associated diseases.

• The sulfated C19 steroid hormones, but not the non-sulfated forms or the sulfated C21 steroids, may extend lifespan and protect against aging-associated proteotoxicity.

Conclusion

• The concept was supported by results from animal models of Parkinson's disease, Huntington's disease and Alzheimer's disease.