An Overview of Sulfur-Containing Compounds Originating from Natural Metabolites: Lanthionine Ketimine and its Analogues

Graphical Abstract

Introduction

For thousands of years, human beings have been trying to identify natural products that treat disease. Innovation in all fields of science and technology has provided scientists the ability to determine the unique and specific chemical compounds, from natural sources, that give rise to the biochemical attributes of these natural products. New advances in extraction and isolation methods provide a very efficient way to utilize the natural products in drug discovery and development [1–8]. Once a natural chemical compound is isolated and purified, subsequent studies are performed to determine the precise interaction between the natural compound and its biochemical target. This, in turn, provides the drug development team an opportunity to modify the molecule in order to improve potency, selectivity and drug like properties.

One example of a natural compound that has found traction in the fields of biochemistry and neuroscience research is lanthionine ketimine (LK, Figure 1). Although historically named as the ketimine, LK exists, primarily as the enamine form at equilibrium. Therefore, a more accurate name of the molecule is lanthionine ketenamine which is still abbreviated LK. Cell penetrating analogs of LK reportedly reduce signs and symptoms of neurological disease and injury in a variety of animal models. Although the biological target of LK remains uncharacterized, neuroprotective activity of LK, and its more cell permeable derivative, LKE, has been observed in models of Alzheimer’s disease [9]; amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) [10, 11] and stroke [12]. Modifications to the structure of LK have provided potential drug candidates for possible treatment of neurodegenerative diseases [13]. This review will cover the detection, biochemistry and related drug discovery efforts associated with LK, its analogues and related cyclic ketimine derivatives.

Figure 1.

The imine and enamine forms of LK

The History of Sulfur-Containing Cyclic Ketimines

As with most natural products and their metabolites, the history of the sulfur-containing cyclic ketimines fortuitously got its start in the lab of Cavallini, when he and is coworkers were studying molecules excreted by cystathioninuric patients [14]. The meticulous study of these compounds and the need for a confirmatory set of chemical compounds led to the group to investigate oxidation products of L-cystathionine. In doing so, Ricci and co-workers determined, in humans, the occurrence of secondary metabolic routes of cystathionine, centered on the production of cystathionine ketimine (CK), which is revealed by the lack of cystathionase in cystathioninurics [14]. Once identified as biomarkers of disease, the Cavallini lab set out to study, in detail, the relevance and fate of these, and other, sulfur-containing cyclic ketimines in human biology. In 1985, Cavallini and colleagues first identified cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid (TDMA), which are the reduced forms of CK and lanthionine ketimine (LK), respectively, in extracts from bovine brain [15]. Later, in 1987, the same group reported finding these molecules in human urine samples [16]. Upon continued research of these sulfur containing natural products, the group subsequently reported that unsaturated and saturated CK and LK can react with phenyl isothiocyanate to afford the respective phenylthiohydantoin (PTH) derivatives with significantly different UV-Vis absorption maxima [17, 18]. At this time, these derivatization and identification techniques were considered state-of-the-art for the quantification of these compounds from biological matrices [17, 18]. Utilizing these methods, in 1988, Cavallini’s group was the first to report the detection of LK in bovine brain [19]. After this discovery, in 1990, the same group reported the detection of CK and LK in human urine and subsequently studied the influence that diet had on the urinary concentration of these compounds [20]. When comparing urine from subjects on non-vegetarian diets, versus humans on vegetarian diets, levels of urinary CK were shown to be lower on the vegetarian diet compared to those obtained on the non-vegetarian diet. When on a vegetarian diet, a person consumes less methionine, a major biological precursor to CK, while levels of cysteine and serine, the proposed precursors to LK, are abundant in vegetables [20]. In the same year, the group reported a new high performance liquid chromatography (HPLC) method for the detection of an additional sulfur-containing cyclic ketimine from bovine brain, namely 3,4-dihydro-2H-1,4-thiazine-5-carboxylic acid or aminoethylcysteine ketimine (AECK) [21]. AECK readily reacts with itself to form a dimer, which subsequently rearranges and spontaneously decarboxylates to produce AECK-decarboxylated dimer (AECK-DD) [22]. Under most biological conditions, AECK is only found in the form of AECK-DD. In 1996, AECK-DD was first detected by the Cavallini group in human urine via GC-MS after functionalization with phenyl isothiocyanate (PITC) [23]. Using the same method, CK and LK were first detected in human brain tissue in 1997 [24]. Later in 1998 and 1999, AECK-DD was reported to be detected in bovine cerebellum and human plasma, respectively [25, 26]. In 2003, an HPLC-electrochemical detection method was developed for the detection of AECK-DD with higher sensitivity than the gas chromatography procedure used previously [27]. In 2009, Pinto, et al. detected AECK-DD in cysteamine pretreated rat brains, at a very low concentrations, using a sensitive HPLC method with electrochemical detection [28]. A few years later, a method was developed by Tsikas et al., which utilized a stable isotope labelled form of AECK-DD as an internal standard to optimize a highly sensitive GC-MS/MS assay for the detection of AECK-DD. This assay was used to detect very low levels of AECK-DD in mouse brain, rat brain and shallots. Using this method, the concentrations of AECK-DD in human plasma and urine were also updated [29, 30]. The ketimine compounds detected in different tissues, and their concentrations, are summarized in Table 1.

Table 1.

Ketimine compounds detected in specific tissues and their corresponding concentrations.

Ketimine Compound	Sample	Concentration
TDMA	Human urine	0.21–1.8 μmol/g creatinine
Cyclothionine	Human urine	0.56–2.0 μmol/g creatinine
LK	Human urine	0.53–2.2 μmol/g creatinine
LK	Human brain	1.1 ± 0.3 nmol/g tissue
CK	Human urine	1.6–12 μmol/g creatinine
CK	Human brain	2.3 ± 0.8 nmol/g tissue
AECK	Bovine cerebellum	0.003 μmol/g wet weight
AECK-DD	Human plasma	4 nmol/L
AECK-DD	Human urine	0.00088–0.052 μmol/g creatinine
AECK-DD	Rat brain	0.008 nmol/g tissue
AECK-DD	Bovine cerebellum	0.6–1.0 nmol/g wet weight
AECK-DD	Shallots	6.8 pmol/g fresh tissue

Metabolism Pathways of Sulfur-Containing Cyclic Ketimines

Two of the sulfur-containing cyclic ketimines described thus far, LK and CK, are recognized as metabolites arising as offshoots from the transsulfuration pathway (TSP) starting with homocysteine [31]. The TSP is involved in many important biological functions including the synthesis of the essential amino acid cysteine [32], the production of the gastrotransmitter, hydrogen sulfide (H2S) [33] and the production of CK [34], for example. Another cyclic ketimine, AECK, shares the same metabolic pathway as CK and LK, but utilizing an alternate starting material, namely cysteamine (Figure 2, [31]).

Figure 2.

Formation of sulfur-containing cyclic ketimines sharing the same metabolic pathway; CβS - cystathionine-β-synthase; GTK - glutamine transaminase K (Modified from K. Hensley, T.T. Denton, Alternative functions of the brain transsulfuration pathway represent an underappreciated aspect of brain redox biochemistry with significant potential for therapeutic engagement [31])

During the methionine cycle, L-methionine is converted to S-adenosyl-L-methionine (SAM) via methionine adenosyltransferase, which later transfers its activated methyl group to an acceptor molecule, such as a protein – a form of protein post-translational modification, DNA or a small molecule, via a SAM-dependent methyltransferase, to afford S-adenosyl-L-homocysteine. S-Adenosyl-L-homocysteine is then hydrolyzed by S-adenosyl-L-homocysteine hydrolase to homocysteine [35]. Homocysteine itself is non-proteinogenic amino acid which is toxic within the central nervous system [36]. One major route of L-homocysteine metabolism begins with the enzyme cystathionine-β-synthase (CβS). CβS catalyzes a condensation between one molecule of homocysteine with either one molecule of serine, where water is eliminated, or one molecule of cysteine, where hydrogen sulfide is eliminated, to yield L-cystathionine (Figure 2) [37]. L-Cystathionine content is elevated in mammalian brain, especially in tumoral forms of the nervous system, such as neuroblastoma, relative to other parts of the body [38, 39]. Currently, it is believed that the major biological fate of L-cystathionine is its cystathionine-γ-lyase (CγL)-catalyzed conversion to cysteine, α-ketobutyrate and ammonia [40–42]. However, CγL is not the only enzyme to metabolize L-cystathionine. L-Cystathionine has been characterized as a substrate for the pyridoxal 5′-phosphate (PLP)-dependent enzyme glutamine transaminase K (GTK), also known as kynurenine aminotransferase-1 (KAT-1) [43–46]. GTK catalyzes the transamination of numerous amino acid substrates, including L-cystathionine, L-lanthionine and L-thialysine, with a variety of α-keto acid substrates, to afford the corresponding α-ketoacids (Figure 2) [47–50]. These α-keto acids rapidly undergo an intramolecular cyclization and subsequent dehydration to afford cyclic ketimines or ketenamines that have been shown to be present in the mammalian brain. In the case of the cyclized α-keto acid derived from L-cystathionine, the historical representation is that of the ketimine, and since the structure has not been confirmed spectroscopically, it is represented in the ketimine form in Figure 2. In the case of L-lanthionine, and its phosphonate derivatives, the imine rapidly tautomerizes to the enamine, which has been thoroughly characterized by 1- and 2-dimensional NMR spectroscopy [51]. In the case of L-thialysine, AECK, the sulfur analog of 3,4,5,6-tetrahydropyridine-2-carboxylic acid (an intermediate in the conversion of lysine to pipecolic acid) is produced [52–55]. It has been hypothesized that AECK is a normal constituent of the mammalian brain and likely plays a role in the modulation of GABAergic neurotransmission [56–60]. This quantifiable, but short lived, cyclic ketimine reacts with a second molecule of AECK followed by a rapid cyclization and decarboxylation to afford the stable aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) [22]. Oxidation at the α-carbon of the sulfur-containing amino acids has been shown to be catalyzed by the enzyme L-amino acid oxidase (AAO), but the transaminase reactions predominate in the brain [43]. There is evidence showing that the ketimines derived from sulfur amino acids are good substrates of an NAD(P)H-dependent reductase found in several mammalian organs, including bovine kidney and brain [61, 62]. The reduction products from LK, CK and AECK are TMDA, cyclothionine and TMA, respectively.

Antioxidant Properties of AECK-DD, CK and LK

In 1961, several years before Cavallini and associates detected AECK-DD in human urine, AECK-DD was prepared by Hermann et al using an enzyme free, organic synthesis method [63]. In 1991, once it was determined that AECK spontaneously forms AECK-DD, a number of studies were initiated to study the biological properties of AECK-DD. AECK-DD reacts with hydrogen peroxide to form taurine and cysteic acid, which are both readily quantitated by chromatographic techniques [64]. Later, the same group identified at least four different hydrogen peroxide mediated AECK-DD oxidation products, with the major product being AECK-DD-sulfoxide [65]. AECK-DD was also determined to be a mitochondrial respiratory chain inhibitor by impeding ADP-dependent oxidation of NAD⁺-linked substrates and by blocking the electron transfer from NADH to oxygen in a concentration dependent manner [66]. The same research group has shown that AECK-DD protects microsomes from L-DOPA induced lipid peroxidation in the presence of an Fe(III)-ADP complex [67]. These researchers have also shown that this molecule protects deoxyribose from degradation induced by an Fe(II)-H₂O₂ complex [67]. In human monocytic U937 cells, AECK-DD was shown to protect against tert-BuOOH-induced oxidative injury at concentrations ranging from 4 to 100 μM [68]. Additionally, mice fed an AECK-DD supplemented diet showed a higher plasma antioxidant potential as measured by the 2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay [69]. All the above studies indicate AECK-DD to be a scavenger of cellular reactive oxygen species (ROS).

Besides being a ROS scavenger, AECK-DD has been shown to possess activity against reactive nitrogen species (RNS). Peroxynitrite is a typical RNS which has been reported to oxidatively modify low-density lipoproteins (LDL) [70] and inactivate α₁-antiproteinase by tyrosine nitration [71]. When LDL was pre-treated with AECK-DD, peroxynitrite-mediated LDL oxidation decreased significantly and the inactivation of α1-antiproteinase, by tyrosine nitration, was also reduced [72]. These studies, spanning the last 28 years (or 58 years if one includes the original synthesis by Hermann and colleagues in 1961), have shown that AECK-DD is a versatile antioxidant with properties as a scavenger for both ROS and RNS. The related compounds CK and LK also have antioxidant, or ROS scavenging, properties. CK is oxidized by hydrogen peroxide to produce a cyclic compound, characterized as perhydro-3-oxo-1,4-thiazapine-5-carboxylic acid (oxo-HTZC), while LK is oxidized to produce 2-oxothiomorpholine-6-carboxylic acid (oxo-TMC) [73]. Many diseases, including neurodegenerative diseases, are characterized by abnormal oxidative stress levels and the release of ROS and RNS. As noted in the next chapter, LK has been more deeply studied for its biological properties and has emerged as a compound with potential as a drug lead to be developed as a potential treatment for multiple neurodegenerative disorders and, as of now, AECK-DD and CK have not. With their chemical similarities to LK, as well as their competitive antioxidant properties, these compounds are biologically relevant analogues of LK and should be examined more thoroughly to determine their potential as anti-neurodegenerative disease treatments, as is being done for LK.

Neuroprotective activity of LK and its synthetic prodrug LKE

Although the exact biological target of LK has yet to be determined, demonstration of the antioxidant capabilities of LK encouraged a renewed interest in its biochemistry. In 2007, Hensley and colleagues reiterated the biological relevance of LK [74]. Following this in 2010, Hensley’s group produced an ethyl ester, prodrug version of LK (LKE) and subsequently determined LKE to have neuroprotective activity [75]. LKE provided classical antioxidant protection of mouse embryonic cortical neurons exposed to hydrogen peroxide or t-butyl hydroperoxide [12], consistent with sulfur-dependent ROS scavenging action. Other biological activities that were not purely chemical in nature, were also observed. For instance, LK limited cytokine-stimulated expression of inducible nitric oxide synthase (iNOS) in cultured EOC-20 microglia, while LKE had a similar effect, but was more potent, presumably due to the improved cell penetrating capability of the pro-drug [76]. In the same studies, LKE protected NSC-34 motor neuron-like cells from toxicity resulting from hydrogen peroxide treatment and from the toxicity of microglia-conditioned medium, confirming the protection of cells from causes of oxidative stress [77]. LKE also promoted growth factor-stimulated outgrowth of neurites in NSC-34 cells and significantly increased the mean neurite length of dissociated chick dorsal root ganglia neurons, indicating LKE’s neurotrophic activity [77]. To determine if LKE provides non-antioxidative neuroprotection, evaluation of the production of native beta amyloid [Aβ_(1–40)] aggregates in SH-SY5Y neuron-like cells was performed [9]. LKE significantly decreased the accumulation of this Alzheimer’s disease related protein aggregate in these cells. Taken together, the data would seem to indicate that LKE exerts beneficial potencies upon multiple types of brain cells and not only through direct chemical antioxidant activity.

In rodent studies, LKE was found to be orally bioavailable and well tolerated. LKE has shown potent protective activities in multiple disease models [9–12, 78–81]. In the SOD1^G93A mouse model of amyotrophic lateral sclerosis (ALS), LKE improved motor function and was able to slow the progression of the disease and increase overall mouse survival [10, 11]. LKE was shown to decrease infarct volume and improve functional recovery after permanent middle cerebral artery occlusion (p-MCAO) in mice [12] as well as decrease the growth of a C6 glioma tumor after xenograft into rat cortices [78]. In the 3xTg-AD mouse model of Alzheimer’s disease (AD), LKE decreased microglial activation, amyloid burden, phospho-tau accumulation and slowed cognitive decline [9]. Recently, in an okadaic acid-induced cognitive impairment zebrafish model of AD, LKE treatment protected the zebrafish against cognitive impairments, reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor and increased phospho-activation of several pro-survival factors [80]. In an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, LKE was able to significantly reduce the clinical signs of MS, compared to control mice [79]. In the same study, LKE decreased IFNγ production in splenic T cells in a dose-dependent manner, while having no effect on IL-17 production, suggesting the protective effects of LKE take place within the central nervous system (CNS) [79]. LKE has also recently been shown to promote locomotor recovery after spinal cord injury by reducing neuroinflammation and promoting axon growth [81]. Table 2 summarizes the pre-clinical models in which LKE demonstrated protective activity.

Table 2.

Neuroprotective Effect of LKE in Different Disease Models

Disease	Model	Activity	References
ALS	SOD1G93A mice	Improves motor function, slows disease progression, increases overall survival	[10, 11]
Stroke	p-MCAO in mice	Lowers stroke lesions, improved motor functions, improved neurologic deficit scores	[12]
Glioma	Tumor xenograft in rats	Slows tumor growth	[78]
AD	3xTg-AD mice	Decreases microglial activation, reduces amyloid burden and phosphorylated-tau accumulation inside neurons, slows cognitive decline	[9]
MS	EAE mice	Reduces clinical signs of MS, decreases IFNγ production in splenic T cells	[79]
AD	Okadaic acid treated zebra fish	Protects against cognitive impairments, reduces brain cell apoptosis, increases BDNF, pAkt and pCREB	[80]
Spinal Cord Injury	female mice	promotes locomotor recovery after spinal cord injury by reducing neuroinflammation and promoting axon growth	[81]

Even with the successful treatment of a variety of in vivo and in vitro models of neurological diseases and disorders, the development of LKE into a clinical candidate has been slowed by the lack of a clear biological target. However, it has been recently reported that LKE has autophagy stimulating properties, which may account for its neuroprotective properties [82]. Autophagy is a vital cellular function responsible for the disposal of cytosolic waste such as misfolded proteins and dysfunctional organelles. Autophagy also supplements cells with energy under starvation conditions [83, 84]. In many neurodegenerative diseases, impaired cellular autophagy is thought to be a major causative factor [85]. In Alzheimer’s disease models, autophagy is thought to digest toxic Aβ oligomers before they have had time to associate into plaques [86]. For example, in a transgenic mouse model of mutant amyloid precursor protein expression, increased autophagy attenuated Aβ pathology as well as cognitive decline [87]. Thus, with the potential of autophagy stimulation as a treatment option for neurological disorders, the use of brain-penetrable, drug-like small molecule autophagy stimulators as treatments for these disorders is possible. Therefore, LKE itself is a good candidate as a treatment option for these and related maladies.

Exploratory Efforts for LK Target Identification

When LK was first detected in bovine brain, it was determined to have a high-affinity binding site in synaptosomal membranes [88]. [³⁵S]-labeled LK was displaceable by AECK and CK, suggesting that LK has a potential functional receptor and that the cyclic-ketimine pharmacophore has a general role in the CNS [88].

To identify bovine brain protein binding partners of LKs, LK was chemically linked to a diaminodipropyl amine (DADPA) functionalized agarose matrix using a modified Mannich reaction followed by treatment with a cell lysate from bovine brain. After elution of the bound proteins by 100 mM LK, proteomic analysis was performed using mass spectrometry-assisted protein microsequencing. Three proteins, collapsing response mediator protein-2 (CRMP2), syntaxin-binding protein-1 (STXBP1) and lanthionine synthetase-like protein-1 (LanCL1), were shown to be enriched by interaction with the solid supported LK [77]. As detailed below, CRMP2, STXBP1 and LanCL1 all play key roles in neurite outgrowth, synaptogenesis and neurotransmission.

CRMP2 is a protein crucial for mediation of growth factor-dependent axon and dendrite development which regulates neuron polarization (the process by which one neurite becomes an axon) during embryonic neurogenesis [89]. The interaction between LK and CRMP2 was further elaborated by Feinstein and colleagues in collaboration with the Hensley group [79]. They found that EAE mice had significant neurodegeneration in both the optic nerve and spinal cord, which was reduced in the LKE-treated mice and that the effects of LKE were associated with a reduced relative level of phosphorylated CRMP2 to CRMP2. The Feinstein group also showed that, in vitro, LKE treatment of oligodendrocyte progenitor cells induces process growth and, in vivo, LKE treatment provides benefit in the EAE mouse model of multiple sclerosis [90]. The facts that LKE treatment is beneficial in AD models, and that CRMP2 has overlapping function with tau proteins that directly form diagnostic neurofibrillary tangles (NFTs) [91, 92] implicated in AD pathology, provide evidence that CRMP2 is a potential biological target of LK.

A second protein identified by the LK-solid-supported proteomics experiment, STXBP1 is part of the presynaptic protein machinery that regulates fusion of neurotransmitter vesicles with the plasma membrane during neurotransmitter docking and exocytosis [93]. Although there is evidence for the direct binding of LK to STXBP1, little work has been done to characterize the specific interactions between this protein and LK.

The third protein identified in the LK-solid-supported proteomics experiment, LanCL1, has been shown by Vanderdonk and colleagues not to be responsible for the biochemical synthesis of LK [94]. However, this does not invalidate LanCL1 as a biological target of LK. LanCL1 was found to bind to CβS and negatively regulate classic transsulfuration in neurons [95]. Overexpression of LanCL1 leads to nerve growth factor-dependent neurite extension in PC12 cells [96] and LanCL1 knock-out mice experience severe neurodegeneration in their early adult period [97]. Although these experiments are not sufficient to confirm LanCL1 as the biological target of LK, they certainly establish a link between LK and LanCL1. Further experiments must be done to reveal the relationship between LK and LanCL1.

Even though this study provided three possible protein binding partners for LK [77], the potential biological target is not limited to these three possibilities. There are numerous limitations to a small molecule, such as LK, being bound to a solid support for use in pulling down interacting proteins. When the small molecule is tethered to the solid support, the active region of the small molecule may inadvertently be used to anchor the small molecule to the solid support, thereby making it unavailable to engage with the target protein. Our research group is currently working on alternate methods of target identification for LK and LKE (unpublished).

The Synthesis of Phosphonate Analogues of LK and LKE

Even though the exact target of LKE remains unclear, the in vitro and in vivo outcomes obtained thus far, provide sufficient rational for an academic lab, or a drug development company, to undertake a thorough structure activity relationship analysis, directed at designing a brain-penetrable, drug-like, small molecule treatment for neurodegenerative diseases, using LK and LKE as lead compounds.

To become a viable drug for the treatment of a neurodegenerative diseases, a small molecule must possess certain properties including its ability to cross the blood brain barrier (BBB). The BBB is a highly selective permeability barrier that separates the circulating blood from the brain extracellular fluid in the central nervous system (CNS). The BBB is formed by brain endothelial cells, which are connected by tight junctions [98]. Generally, hydrophilic substances are not able to diffuse across the BBB and are excluded from entering the brain. The BBB contains a limited number of conveyance mechanisms for hydrophilic compounds such as transport through ion channels, receptor-mediated transcytosis, adsorptive-mediated transcytosis and carrier mediated transport [99]. The hydrophobic nature of the BBB endothelial cell membranes only allows for passive diffusion of lipophilic small molecules [100] and stringent requirements must be followed to create a small molecule capable of passively crossing the BBB and most CNS drug discovery efforts focus on this requirement [101]. In 1997, Lipinski and colleagues developed a set of rules for a small molecule to be orally bioavailable [102]. Although these rules were not directly created to evaluate the ability of a drug to cross the BBB, the rules are translatable to the BBB since they were developed as a guide to designing drugs that passively diffuse through a cell membrane. With these characteristics in mind, using LK and LKE as starting points, our research group recently developed a set of phosphonate analogues of LK, LK(E)-P(E)s (lanthionine ketenamine (ester)-phosphonate (ester)s, Figure 3) [13]. In order to compensate for the increased polarity of the phosphonic acid group, relative to the carboxylic acid group, at the C-3 position, alkyl substituents with various degrees of lipophilicity were installed at C-2. Of the compounds in this set tested thus far, the synthesized phosphonates are more potent autophagy stimulators than the lead compound LKE [13]. Research with the LK(E)-P(E)s in drosophila melanogaster models of neurodegenerative diseases is ongoing and will be reported in due course.

Figure 3.

Synthesized LK phosphonate analogues [13]. The phosphonic acid residue affords a highly hydrophilic group, which is balanced by the inclusion of hydrophobic groups at the 2-position and the ethyl ester group

Conclusion and Outlook

In conclusion, the sulfur containing cyclic ketimines: LK, CK, AECK and AECK-DD are now considered to be natural compounds with important biochemical properties. Over the past 20 years, numerous techniques have been developed and deployed, such as HPLC-UV, HPLC-ECD, HPLC-MS, GC, GC-MS, GC-MS/MS to identify and quantify each cyclic ketimine, or ketenamine, from multiple different sources, such as human, bovine and rat brain, bovine kidney, human and bovine plasma, human urine and vegetables. The biochemical genesis of all these cyclic ketimines and ketenamines have a common step where CβS catalyzes the reaction between two sulfhydryl groups, to eliminate hydrogen sulfide, or a sulfhydryl group and an alcohol group, to eliminate water, to afford substrates for the widely expressed glutamine transaminase, GTK.

Although the natural biological functions of these cyclic ketimines remain to be fully elucidated, each of these molecules, ex-vivo, has been shown to have a number of protective biochemical functions, such as antioxidant properties, anti-neurodegenerative activities and autophagy stimulation. AECK-DD exhibits robust activity against ROS and RNS in vitro, LK, and its synthetic prodrug LKE, show not only antioxidant properties, but also provide neuroprotection in multiple pre-clinical models of neurodegenerative diseases. LK, LKE and more recent analogues of LKE (i.e., LKE-phosphonates), have autophagy stimulation properties which may underlie their positive biochemical attributes. In conclusion, these natural, sulfur-containing cyclic ketimines provide a solid foundation for future medicinal chemical campaigns directed towards the development of small molecules for the treatment of neurodegenerative diseases.

Highlights.

• Lanthionine ketenamine ester relieves neurodegenerative phenotypes in mouse models of amyotrophic lateral sclerosis, Alzheimer’s disease and multiple sclerosis.

• Lanthionine ketenamine ester-phosphonates stimulate autophagy.

• Sulfur-containing cyclic ketimines and ketenamines are natural products with antioxidant and neuroprotective activities.

• Drugs for the treatment of Alzheimer’s disease, Amyotrophic Lateral Sclerosis and Parkinson’s Disease may be developed from lanthionine ketenamine-phosphonates.

Abbreviations

Aβ

beta-amyloid

ABTS

2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid

AD

Alzheimer’s disease

AECK

aminoethylcysteine ketimine

AECK-DD

aminoethylcysteine ketimine decarboxylated dimer

ALS-FTD

amyotrophic lateral sclerosis-frontotemporal dementia

BBB

blood brain barrier

CβS

cystathionine-β-synthase

CγL

cystathionine-γ-lyase

CK

cystathionine ketimine

CNS

central nervous system

CRMP2

collapsing response mediator protein-2

DADPA

diaminodipropyl amine

ECD

electrochemical detection

EAE

experimental autoimmune encephalomyelitis

GC

gas chromatography

GTK

glutamine transaminase K

HPLC

high performance liquid chromatography

oxo-HTZC

perhydro-3-oxo-1,4-thiazapine-5-carboxylic acid

KAT-1

kynurenine aminotransferase-1

LanCL1

lanthionine synthetase-like protein-1

LDL

low-density lipoproteins

LK

lanthionine ketimine, lanthionine ketenamine

LKE

lanthionine ketimine ethyl ester, lanthionine ketenamine ethyl ester

LKE-P

lanthionine ketenamine ester-phosphonate

LK(E)-P(E)s

lanthionine ketenamine (ester)-phosphonate (ester)s

MS

mass spectrometry

MS/MS

tandem mass spectrometry

p-MCAO

permanent middle cerebral artery occlusion

NFTs

neurofibrillary tangles

Oxo-HTZC

perhydro-3-oxo-1,4-thiazapine-5-carboxylic acid

Oxo-TMC

2-oxothiomorpholine-6-carboxylic acid

PITC

phenyl isothiocyanate

PLP

pyridoxal phosphate

PTH

phenylthiohydantoin

RNS

reactive nitrogen species

ROS

reactive oxygen species

SAM

S-adenosyl-L-methionine

STXBP1

syntaxin-binding protein-1

TDMA

1,4-thiomorpholine-3,5-dicarboxylic acid

oxo-TMC

2-oxothiomorpholine-6-carboxylic acid

TSP

transsulfuration pathway