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Theoretical Discussion of the Probable Mechanisms for the Antiviral Effects of L-Lysine

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Abstract

We present a theoretical discussion of L-Lysine as a universal treatment of viral diseases, including COVID19. This hypothesis is based on a widely published scientific literature on replication inhibition effects of a high lysine/arginine concentration ratio at many critical points of the viral life cycle. We further speculate that the body of evidence we reference may lead to a novel approach to developing universal synthetic vaccines against SARS-CoV-2 and the entire Coronavirus family as well as against other viral families.
Theoretical Discussion of the Probable Mechanisms for the Antiviral
Effects of L-Lysine
Rony Tal, Christopher Kagan, Bo Karlicki, Alex Chaihorsky
Abstract
We present a theoretical discussion of L-Lysine as a universal treatment of viral
diseases, including COVID19. This hypothesis is based on a widely published scientific
literature on replication inhibition effects of a high lysine/arginine concentration ratio at
many critical points of the viral life cycle. We further speculate that the body of evidence
we reference may lead to a novel approach to developing universal synthetic vaccines
against SARS-CoV-2 and the entire Coronaviridae as well as against other viral
families.
L-Lysine is a naturally occurring basic amino acid (14). Our recent finding that it is
effective against chikungunya (30), a viral disease transmitted by mosquitoes, adds to
its known effectiveness against herpes, and SARS-CoV-2 as reported by our group (2)
(3) (4). It is also effective against adenovirus type 1, SV 40, polyoma, cytomegalovirus,
measles, and Marek’s disease in chickens (5). Kagan, a co-author of this paper,
published the initial report of the clinical use of L-Lysine against herpes type 1 and 2 in
Lancet in 1974 (1). In vitro evidence by Tankersley (6) and Griffith (2) (6) supports the in
vivo studies, and Griffith and Kagan elucidated the then current understanding of the
mechanisms of action.
This paper expands our understanding of the mechanisms of action and discusses the
probability that we have in L-Lysine an example of a universal treatment; one that is
effective against many of the viruses within a given viral family. We have demonstrated
that L-Lysine is an effective treatment against more than one viral family (2) (3) (30). It is
critical to develop such treatments during the COVID19 pandemic particularly if it works
against the entire coronavirus family. We will present our theory regarding potential
mechanisms that explain why it is likely to be a universal treatment, and speculate on
some of the reasons why from both an evolutionary and a basic science perspective.
Just as humans, gorillas, chimpanzees and orangutans have a common ancestor that
likely resembled a baby gibbon (8), viruses must also have early common ancestors
that evolved to use amino acids based on their availability. Such is probably the case
involving the two amino acids lysine and arginine which are similar in structure. Arginine
is one of the non-essential amino acids since it is synthesized primarily in the
intestinal-renal axis, and thus it would always be available. Lysine is not always
available because it is an essential amino acid, and its supply depends predominantly
on dietary intake. We suspect that most viral families evolved to use arginine, which
makes sense given the aforementioned constraints. The one exception is the retrovirus,
HIV, where lysine seems to stimulate growth rather than repress it as is the case in
other classes of viruses (9).
Lysine affects a wide range of physiological phenomena summarized in
https://www.empowerpharmacy.com/drugs/l-lysine-hcl-injection#footnote10_p2g3b47 :
which is paraphrased as follows: Effects include LDL receptor docking caused by
lysine-rich regions in LDL particles. Nascent mammalian cytoplasmic proteins have
specific lysine-containing motifs present at carboxyl groups which act as identifying tags
allowing them to be retained in the endoplasmic reticulum. Titin is a large sized muscle
protein which appears to control elasticity of different types of muscle. At its center, it
contains a lysine-rich motif, PEVK (proline, glutamate, valine, and lysine) which is
believed to provide the required elasticity acting as a "spring”. Lysine is stored in muscle
tissues where it may act as a binding agent besides playing an important role in
collagen formation and wound healing. Lysine competes with other dibasic amino acids
like arginine and ornithine for transportation not only in intestinal mucosa and renal
tubules but also in mitochondria. It also antagonizes arginine by way of the following
three mechanisms:
1. Lysine functions as an antimetabolite of arginine.
2. Lysine competes with arginine for reabsorption in the renal tubules thereby
increasing arginine excretion in urine.
3. If there is an excess lysine in the gut, absorption of arginine decreases.
There is evidence, some circumstantial, that illuminates the undeniable central role of
the lysine/arginine interchangeability and opposition. We demonstrate lysine’s
significant therapeutic and preventive influences in six separate loci:
1. In and around the Receptor Binding Domain (RBD) and the S-protein of
SARS-CoV-2.
2. In the ACE2 receptor, and other components of the cell membrane.
3. Affecting arginine metabolism and generation of nitric oxide (NO) intracellularly.
4. Affecting viral trafficking through intracellular compartments, assembly of viral
particles and their release from the cell.
5. The effect of methylated lysine on host immunology.
6. Rare arginine codons: exchange of lysine for arginine during protein synthesis.
It is intriguing that lysine and arginine are interchangeable according to
the Circular Genetic Code arrangement proposed by F. Castro Chavez and others (7).
Lysine and arginine as major protein residues are involved in cation π interactions
between protein/protein and protein/nucleic acid interactions (11). In hundreds of
available protein/protein crystallographic studies, arginine has a significantly higher
electrostatic "influence" than lysine and histidine in interacting with other amino acids.
Considering that the interaction of the Receptor Binding Domain (RBD) to ACE2
involves energetically dynamic contacts between clouds of electrons, the biological
quantum mechanical context is extremely difficult, if not impossible, to calculate
accurately. For example, it is probably not possible to make precise predictions about
how known mutations in the SARS-CoV-2 genome affect the interaction between ACE2
and the RBD. Nevertheless, we know that the arginine/lysine concentration ratio is a
key determinant of the strength of the interaction since lysine has positive effects in
COVID19 treatment and prevention (3) (4).
It is essential that lysine treatment must be accompanied by maintaining a strict low
arginine and coffee free diet. Coffee (caffeine) inhibits arginase (34), a key enzyme of
arginine catabolism which further illustrates arginine’s central role in the life cycle of
SARS-CoV-2 and in other viruses.
Besides being used in protein synthesis, arginine serves another key role as a substrate
in nitric oxide (NO) generation in mammals (12). NO plays a dual role in inflammation.
On the one hand, NO can kill microorganisms and has a protective effect on the body,
helping fight against various viruses. On the other hand, NO can damage normal tissue,
such as liver cells, to generate pathogenic effects (28).
Lysine has been shown to block arginine transport in vitro (31), most likely via a
competitive inhibition and down regulation of cationic amino acid membrane
transporters. See arginine metabolism figure here:
https://academic.oup.com/view-large/figure/163362835/1662Sfig1.jpeg (13).
We hypothesize that in general high lysine/arginine ratios will reduce NO production and
prevent its pathogenic effects.
Lysine, being a NO inhibitor, may also play a role in one of modern medicine's greatest
challenges, the rise of antibiotic resistant bacteria (16). Determining if lysine and
antibiotics are synergistic merits further investigation. Our observations suggest this will
be the case.
NO levels are associated with numerous autoimmune diseases. While many
autoimmune conditions are associated with a lack of endogenously produced NO, some
autoimmune illnesses have excess NO levels. Lysine may be beneficial for those who
have autoimmunity associated with high NO levels because it inhibits arginine
metabolism. Supplements/foods with high arginine and nitrates may alleviate symptoms
of autoimmune diseases associated with low NO. Subjects who have autoimmune
illnesses associated with low NO and have used lysine for COVID19 prophylaxis
reported exacerbated symptoms of headaches, circulation issues and various other
autoimmune symptoms (personal communication from Bo Karlicki). Most of the
autoimmune illnesses associated with low NO levels are found in females (17).
Gender differences in NO production are vast even when corrected for weight (35).
Inducible nitric oxide synthase (iNos) which is the NO production process in
inflammatory signaling causes higher NO overproduction in males. This results in a
positive feedback loop of inflammation and may be the main contributing factor to why
there is a gender disparity in the COVID19 mortality, with male mortality significantly
higher. Conversely, female dysregulation of NO production may cause lower mortality,
but may also be the reason why females suffer a higher ratio of chronic post COVID19
symptoms. These chronic COVID19 symptoms overlap and are easily confused with the
symptoms of autoimmune illnesses. There is mounting data supporting this hypothetical
NO dynamic. Severe COVID19 infections are linked to higher NO levels than controls
(18). Nearly all successful treatments of moderate to severe COVID-19 are NO
inhibitors.
Microclotting is a significant problem in COVID19 including in young patients and those
with long term COVID19 (32). High lysine concentrations have demonstrated a
significant effect on in vitro coagulation time and clinically in COVID19 patients (33).
This mechanism has a potential to yield vast medical benefits in treating the
hypercoagulative nature of COVID19 and other coagulative disorders and illnesses.
Recent sequence data compilation from the CDC (15,19) of all known SARS-CoV-2
family branches have shown an interesting evolution of escape mutations in key
residues in and around the S1-protein. A larger than expected number involves lysine
and/or arginine. These mutations, either combined or singular, such as in the delta
variant, indicate that alternative residues that increase binding to ACE2 have evolved in
the population to escape initial neutralizing antibodies. Some mutations have made the
delta variant 40-60% more transmissible, and it may be more dangerous to children
than previous variants (20).
It is established that two key lysine residues in ACE2 are first to interact with the RBD
(21). Flooding the body with a relatively high local concentration of free lysine
(approximately 100 uM or higher) acts competitively to inhibit binding to some degree,
albeit significant reduction in arginine is also required for inhibition. High concentration
of lysine combined with low arginine concentration may also inhibit arginine interactions
required for cell entry. The furin cleavage site between S1 and S2, RRAR, is encoded in
part by two consecutive rare arginine codons (CGG CGG). It is a unique feature of
SARS-CoV-2 compared to its coronavirus predecessors, and it is involved in a complex
protein/protein interaction with other proteases and glycosylated moieties that lead to
cleavage that depends on this arginine cluster for releasing S1 and S2 properly leading
to cell entry (22). A possible reason for this interference with furin based cleavage may
involve another feature of SARS-CoV-2, namely, rare arginine codons can be
overpowered by a high lysine/arginine concentration ratio because of a low abundance
of tRNA’s for rare codons of arginine. Another molecular mechanism of mistranslation
allows flexibility to replace arginine coded by AGA with lysine using the cognate tRNA
codon for lysine (23). This fact might account for lysine substitutions of key arginines
involved in protein/protein or protein/RNA interactions in viral assembly.
Increasing lysine concentrations significantly above physiological levels may affect the
activity of methylated and acetylated lysines. Acetylated lysine acts specifically to
regulate cellular membrane transport complexes. High naked lysine concentration may
interfere with viral entry and proper acetylation of viral protein trafficking through
intracellular compartments such as the endoplasmic reticulum (ER)/Golgi complex (24).
Recent studies have shown that naked and derivatized lysine may act downstream from
acidified compartments near the cellular membrane closer to the nucleus (25). High
intracellular concentration of naked lysine may compete with this process. Additional
supporting evidence comes from gradient density analysis showing that omission of
lysine from infection study medium prevents the proper intracellular assembly of
reovirus. Only empty virions are formed indicating that acetylated lysines are essential
for proper viral assembly (26). We suspect it will have a similar effect in SARS-CoV-2.
Another example involves distinct lysine methylation/demethylation of histones. It has
been proven to influence downstream chromatin events for expression or repression of
specific genes in large chromosomal regions. This affects, among other things, the
expression of genes involved in regulating immune conversion of M1 type inflammatory
macrophages to M2 type anti-inflammatory macrophages (27). High levels of lysine may
therefore regulate expression of specific genes. This may explain the rapid resolution of
fever following the first administration of lysine to COVID19 patients.
It is possible that high concentrations of lysine may also interfere with internal arginine
rich clusters required for proper viral replication. An example is the discovery of an
arginine rich 28 amino acid long peptide encoded by human cytomegalovirus (HCMV)
(5340-5424) included in the short unique repeats gene region and part of the long
repeat which was used to generate a polyclonal antibody that interacted with the entire
herpes family in ELISA and neutralization experiments (personal communication, Alex
Chaihorsky, patent # US5534258A
(https://patents.google.com/patent/US5534258A/en). This completely synthetic,
algorithmically derived short peptide chain was used successfully as a “micro-antigen”
in a vaccine in chickens against Marek's disease. The same algorithm yielded more
micro-antigens that, together with the abovementioned one, each demonstrated unique
universality both in-vitro and in-vivo, including protection studies against many herpes
viruses both human and animal, including genital herpes, Epstein-Barr, cytomegalovirus ,
varicella-zoster and Kaposi’s sarcoma (see relevant US patents by D.B. Golubev and A.
Chaihorsky, a co-author of this article). We speculate that this peptide vaccine may
target similar arginine rich clusters in all herpes viruses. Other mechanisms of
protection are also possible, but the unique feature of this approach - universality
against many viral members of the same group seems to remain intact. Note that high
lysine/arginine ratio treatment is effective in herpes in general. SARS-CoV-2 also, for
example, contains viral arginine and lysine clusters, localized from 28330-28340 and
29455-29461, respectively, of the N gene encoding the nucleocapsid phosphoprotein
(29). Their critical functions in viral replication may be interfered with by the addition of
high concentrations of lysine. Targeting these clusters as antigens offers hope for
universality in future vaccines against Coronaviridae.
CONCLUSION
Lysine exerts myriad physiological effects and provides antiviral therapeutic
value in the treatment of diseases associated with many viruses. The
widespread effects of lysine on viral replication include its counteracting arginine
metabolism, preventing viral entry and infection, preventing viral intracellular assembly,
and boosting immune anti-inflammatory response. These effects point to the universal
potential of lysine as a therapeutic/preventive measure. It is demonstrably effective and
affordable against viral infections including SARS-CoV-2. It is reasonable to expect
more variants to emerge which makes it critical to have universal therapies.
Lysine appears to operate at multiple stages of the viral infectivity cycle, some
apparently early as shown in our previous work, and at multiple host dependent stages,
from early to late, as shown in this paper. This conclusion is supported by the evidence
here and by inference from its spectrum of success against different viruses and across
viral families. The probability that SARS-CoV-2 mutations will be able to evade its
inhibitory effects decreases as the supportive data that it plays a universal role
increases. It is unlikely that the virus can completely overcome through mutations the
multiple sites where lysine has an inhibitory effect directly on viral replication and on the
host support critical for viral replication.
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Background: Virus replication strongly depends on host metabolic machinery and essential cellular factors, in particular, on amino acid profiles. Amino acids play an important role in the pathogenesis of all virus-related infections both as basic substrates for protein synthesis and as regulators in many metabolic pathways, including gene expression. The inhibitory effects of deficiency or excess of these essential elements on virus replication are widely appreciated. Although the same interrelationship between host cellular factors and HIV have been recognized for a long time, the effects of amino acids on HIV-1 RNA replication dynamic is not yet well documented. Our aim was to determine in this pilot study the direct effect of L-lysine amino acid on HIV-1 RNA replication in vitro in HIV-infected patients. Methods: A total of 100 HIV-1-infected males without highly active antiretroviral therapy (HAART) were monitored in our center. The patients were in stage A of the disease according to the 1993 Centers for Disease Control (CDC) classification system for HIV-infection. Patients with HIV were enrolled in one stage (A) of the disease with the average amount CD4 lymphocytes in the range of 200-300 cells/µL at the time of sample acquisition. For evaluation of the effects of essential L-lysine amino acid on HIV-1 RNA replication level, we used a model of amino acid-excess system in vitro following incubation of plasma samples for 24 h at 25 °C. Quantitative HIV-1 RNA assay was performed using (RT-PCR) reverse-transcriptase polymerase chain reaction (Rotor-Gene Q, QIAGEN, Germany). Results: The mean HIV-1 RNA levels were significantly higher in the enriched peripheral blood mononuclear cells plasma samples HIV-infected subjects after 24 h incubation at 25 °C temperature than in the plasma samples the same patients studied on the date of blood tests (p < 0.0001). The number of HIV-1 RNA copies increased in 1.5 times. We observed that in plasma of the same HIV-infected patients after adding L-lysine and following incubation in vitro, viral load increased significantly in comparison with standard samples (p < 0.0001). The increased viral load was found in 100/92 (92%) of HIV-infected subjects. The average number of HIV-1 RNA copies in samples had increased by 4.0 times. However, we found no difference in HIV-1 RNA levels after replacement of L-lysine for L-arginine in comparison samples in the same HIV-infected patients. It is obvious that the addition of L-arginine does not increase viral replication in vitro as L-lysine amino acid supplement does. Additionally, no increase in viral load was determined after adding L-lysine and non toxic doses of its inhibitor (L-lysine alpha-oxidase) in plasma samples. Conclusions: The results show that L-lysine amino acid excess is characterized by significant increased of HIV-1 RNA copies in enriched peripheral blood mononuclear cells plasma samples of HIV-infected patients. There was evidence for an association between L-lysine supplementation and HIV-1 RNA replication and the level changes of this host essential nutritional element play a key role in the synthesis of the virus proteins and in transcription initiation of the retrovirus life cycle. High intake of L-lysine amino acid may increase the risk of high viral load, subsequent acceleration of immunosuppression and HIV progression. Overall results demonstrate that the simple L-lysine-related model in vitro can be widely used for practical purposes to evaluate HIV-1 RNA replication dynamic, disease prognosis and new approaches in treatment of the patients with human immunodeficiency virus. Although the impact mechanism of L-lysine amino acid on the viral load in the pathogenesis of HIV-infection is at present conjectural and requires further development, the results highlight an interesting target in antiviral therapy, and this statement remains to be proved in further research and clinical trials.
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Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. (a) Marked enhancement by platelet activation; (b) deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; (c) saturability (fibrinogen binding approaches saturation at more than 12 μM, within 10 min; lysine binding at more than 100 mM within 1 min); (d) Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); (e) reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; (f) inhibition by the oligoamines His-Lys and Lys4; (g) inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; (h) specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated; Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.
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In the studies conducted, arginine deficiency suppressed herpes simplex virus replication in tissue culture. Lysine, an analog of arginine, as an antimetabolite, antagonized the viral growth-promoting action of arginine. The in vitro data may be the basis for the observation that patients prone to herpetic lesions and other related viral infections, particularly during periods of stress, should abstain from arginine excess and may also require supplemental lysine in their diet.