HDACs deacetylate histones pathway modulated by Coronavirus
Literature search on PubMed, web of science and scopus, demonstrated a robust and already credited interaction between virus S protein and ACE as host receptor. Our miRNet query these two proteins as key words is illustrated in figure1.
As shown in the figure 1, ACE and Spike miRNet analysis returned Histone Deacetylate (HDAC) pathway modulation strongly significant (p = 0.0113) with miR–335–5p involvement in ACE and spike gene expressions. Furthermore, ACE, AT1R and ACE2 miRNet query returned miR–26b–5p modulation as further information.
From the above bioinformatics results HDAC has a pivotal role in modulating COVID–19 pathogenicity. The importance of this result can be fully appreciated following the comparison with clinical data as illustrated in the clinical comparison paragraph, reported later in this paper.
This is noteworthy information to be exploited for therapy interventions.
In Fig 2 we identified the main mechanisms and interactions surged from our bioinformatics analysis connecting viral spike protein and ACE2 host receptor to HDAC activity.
The inhibition of ACE/ACE2-ATR1-Cholesterol-HDAC axis signals may then result in a decrease of SARS-CoV–2 infection (in red are indicated drugs interfering with this mechanism). More in detail, it is known that ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 converts angiotensin II in the vasodilator angiotensin thus functioning as a negative regulator of the renin-angiotensin system (RAS) system 13. It has also been demonstrated that the binding of the coronavirus spike protein to host protein ACE2 leads to ACE2 down-regulation, which in turn results in excessive accumulation of angiotensin II. Furthermore, it has already been demonstrated that the binding of S protein of COVID–19 to ACE2 affects the balance of rennin-angiotensin system (RAS) where the activation of angiotensin type–1 receptor (AT1R) leads to exacerbation of severe pneumonia 14.
Histone acetyl-transferases (HATs) acetylates conserved lysine amino acids on histone proteins via the transfer of an acetyl group from acetyl-CoA. Once the acetyl groups, added by HATs to the histones, enter the nucleus, they are removed by HDACs and are incorporated into chromatin. This restores the deacetylated form and, at the same time, releases acetyl-CoA which might be used by 3-hydroxy–3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. This is the key enzyme of cholesterol biosynthesis pathway; notably, plasma membrane cholesterol promotes binding of COVID–19 to ACE2, fostering virus entry into the cells.
The analysis on miRNet, using miR–335–5p and miR–26b–5p to look for their common modulated pathways, evidenced that four pathways were significantly modulated by both miRNAs, as indicated in Table 1. Interestingly, HDAC has a central role in many diseases in which these pathways are involved and is representing an applicable target under investigation for new drugs discovery.
Table 1: Pathways modulated by miR-335-5p and miR-26b-5p obtained using miRNet analysis.
Pathways modulated by miR-335-5p and miR-26b-5p
|
P value
|
|
|
Legionellosis
|
0.00143
|
|
|
Steroid biosynthesis
|
0.00254
|
|
|
MAPK signalling pathway
|
0.0197
|
|
|
Cytokine-cytokine receptor interaction
|
0.0201
|
|
|
Histone acetylation plays an important role in the regulation of pro-inflammatory gene expression in L. pneumophila infected lung epithelial cells15. In addition, it has been observed an increase in the number of superinfections from Legionella spp. in SARS 2. Interestingly, the other 3 pathways of table 1 are all involved in steroids/cholesterol metabolism. In patients with chronic obstructive pulmonary disease (COPD), where HDAC2 activity is impaired, the inflammatory response is often steroid-resistant16. For this reason, statins may restore the function and expression of depleted HDAC2 via modulating the mevalonate cascade 17.
The discovery of HDAC inhibitors has proven to be an important tool for the study of HDAC functions and mechanism of actions. Some of them have been developed and well described with the purpose to be used for hematological malignancies in clinical practice 18. Importantly, HDAC inhibitors seem to be effective bronchodilators by enhancing acetylation of substrates other than histones such as HSP20 and cortactin 19. Furthermore, some HDAC inhibitors can block ANG II-induced cardiac hypertrophy. Based on further animal studies, ANG II infusion increases class I HDAC2 activity in the heart. Class I HDAC inhibitors can also attenuate ANG II-induced cardiac fibrosis 20. Apart from their ability to lower cholesterol, statins have been proven to be able to inhibit the activity of HDAC too. Intriguingly, the above effects of statins might also be relevant to the events leading to SARS-CoV–2 pathogenesis 21. The above effects of statins might also be relevant to the events leading to SARS-CoV–2 pathogenesis. Another effect of statins which deserves attention is their capacity to reduce the expression of TLR4 and regulate the TLR4/Myd88 (myeloid differentiation primary response) /NF-κB signaling pathway22. This pathway, due its pro-inflammatory relevance, plays an important role in the severity of respiratory virus infections such as SARS-CoV and MERS-CoV. Indeed, the MYD88 gene was observed to be highly induced by SARS-CoV infection 23. Furthermore, activation of NF-κB downstream of TLR-MYD88 is a hallmark of coronavirus infections, and its inhibition reduced lung infection significantly increasing mouse survival after SARS-CoV infection 24. Interestingly, statins do not affect significantly the MYD88 level under normal conditions, but prevent its increase during hypoxia or after oxidative stress25, i.e. two conditions occurring during severe respiratory dystress. Atorvastatin was also shown to significantly attenuate TLR4-mediated NF-κB activation 26. The combination of these two effects has been proposed to limit the burst of inflammatory cytokines and chemokines characterizing SARS, MERS and SARS-CoV–2 pneumonia 27.
Clinical comparison: Hypertension in COVID–19 patients
To support our hypothesis that antihypertensive drugs may also yield anti-SARS-CoV–2 effects, comparison with clinical data is very important. Indeed, hypertensive patients often undergo therapies that involve the use of ACE-inhibitors and ATR1 antagonists. We believe that these drugs may interfere with COVID–19 clinical course by modulating the mechanisms evidenced above (ACE/ACE2-ATR1-Cholesterol-HDAC axis). In our hypothesis, patients with hypertension using ACE-inhibitors or ATR1 antagonists, have decreased Angiotensin II production or AT1R down-regulation. In both cases HDACs are not stimulated to produce ACE2, reducing its availability for SARS-CoV–2 infection.
To support this hypothesis, we thus analyzed some clinical data retrieved from a previous Chinese study28. In this study, 1099 patients with laboratory-confirmed COVID–19 from hospitals in China (through January 29, 2020) were studied. Primary End Points (admission to an intensive care unit, ICU; the use of mechanical ventilation; or death) and severity of the disease have been evaluated. Coexisting illness were also considered: chronic obstructive pulmonary disease (COPD), Diabetes, Coronary heart disease, Cerebrovascular disease, Hepatitis B infection, Cancer, Chronic renal disease and Immunodeficiency. The authors considered 165 hypertensive patients (124/75% without disease severity and 41/25% with disease severity), 261 without any coexisting illness (194/74% without disease severity and 67/26% with disease severity) and 178 with coexisting illness (123/69% without disease severity and 55/31% with disease severity). In addition, between 165 hypertensive patients, 141/85% were without End Point and 24/15% with End Point; 261 patients without any coexisting illness 222/85% were without End Point and 39/15% with End Point; and 178 patients with coexisting illness 139/78% were without End Point and 39/22% with End Point. These results indicate that patients with hypertension and the one without coexisting illness, have the same severity and End Points. On the other hand, patients with other illness had higher severity and End Points. In particular, COPD patients were the ones with the highest End Points (58%).
The coexisting illnesses considered in the cited study were: chronic obstructive pulmonary disease (COPD), Diabetes, Coronary heart disease, Cerebrovascular disease, Hepatitis B infection, Cancer, Chronic renal disease and Immunodeficiency. On cancer patients there are only limited information, but they deserve more attention based on their immune-compromised condition.
We performed a comparison by chi-square test to evaluate the proportions of severity and proportions of Primary End Point in Hypertension vs patients with Coexisting diseases and in Hypertension vs any Coexisting diseases. As indicated in Table 2, hypertensive SARS-COV–2 infected patients were mainly without disease severity and without Primary End Points. Related to all the other patients of the study (Non hypertension), Hypertension infected patients have the same severity primary End Point than patients without any coexisting disease (data are not significantly different). A favourable trend can also be observed when Hypertension is related to the patients with other coexisting diseases, since hypertensive patients demonstrated an higher number of patients with no primary End Point. Also, hypertensive patients related to non hypertensive infected patients showed a better disease severity and decreased primary End Point.
These results are in favor of our hypothesis that infected COVID–19 hypertensive patients can better support the infection than the other patients with co-existing diseases. This may probably account for the anti-hypertensive drugs treatment performed. Also, patients with hypertension are in line with disease outcome of infected patients without coexisting illness, probably indicating that anti-hypertensive drugs generate similar physiological conditions present in patients that were healthy before COVID–19 infection.
Table 2: Chi-square analysis for the evaluation of the proportions of severity and proportions of Primary End Point in COVID-19 infected patients.
|
Without disease severity
|
With disease severity
|
p-value*
|
Hypertension
|
124
|
41
|
|
Non hypertension
|
802
|
132
|
< 0.001
|
Any coexisting disease
|
194
|
67
|
0.849
|
Coexisting disease
|
123
|
55
|
0.212
|
|
Primary End Point
|
no Primary End Point
|
p-value*
|
Hypertension
|
24
|
141
|
|
Non hypertension
|
43
|
891
|
< 0.001
|
Any coexisting disease
|
39
|
222
|
0.910
|
Coexisting disease
|
39
|
139
|
0.078
|
*comparison with hypertension (chi-square test)
Therapeutics for COVID–19
Recent publications have brought attention to the possible benefit of chloroquine and hydroxychloroquine, broadly used antimalarial drugs, in the treatment of patients infected by the novel emerged coronavirus (SARS-COV–2). Ground-breaking in vitro studies demonstrated their potential efficacy to treat novel coronavirus infection. The first results obtained from more than 100 patients in China showed the superiority of chloroquine as compared with standard best treatment of the control group with regard to reduction of pneumonia severity, duration of symptoms and delay of viral clearance, all in the absence of severe side effects. Similar studies have undertaken in EU and USA in the recent two weeks, and chloroquine and hydroxychloroquine are about to be definitely included in the recommendations regarding the prevention and treatment of COVID–19 pneumonia. The mechanism underlying the antiviral effect of these drugs resides in the abundance of extra nitrogens: once they cross the membrane and enters an organelle, the organelle is prevented from reaching a lower pH. Under alkalinisation conditions, the phagolysosome enzymes cannot work because the donor group will be a hydrogen ion, an event which disables the hydrolysis required for coronavirus replication. Alongside this mechanism, chloroquine has also been reported to cause an under-glycosylation of ACE2. Interestingly, low glycosylation levels of ACE2 strongly reduce the binding affinity of SARS-CoV–2 and consequently its cellular entry. Hence, the anti-malarial agents are supposed to have a twofold anti-SARS-COV and SARS-Cov–2 viruses: a direct virostatic one which inhibits their replication via phagolysosome alklynization, and a preventive one based on the reduced capacity to bind COVs cellular receptor. Interestingly, this latter effect of RAS antagonists may be considered as coherent with the above-supposed for HDAC-inhibitors.
Accelerated Phase–2clinical trials have been approved in Italy and in USA to evaluate the efficacy of tocilzumab in COVID–19 pneumonia patients. Three hundred and thirty patients are enrolled in each of the studies, whose primary endpoint is to determine the one month mortality rate 29,30.
Tocilizumab is a humanized monoclonal antibody that acts as an IL–6 receptor antagonist currently approved as a biological agent to treat rheumatoid arthritis, systemic juvenile idiopathic arthritis, juvenile idiopathic polyarthritis and giant cell arteritis in adults 31. Tocilzumab is also under evaluation for the treatment of a variety of autoimmune diseases. The wide spectrum of clinincal and preclinical applications of tocilizumab find its fundament in its pharmacological target: indeed, IL–6 is a pleiotropic pro-inflammatory master player cytokine. IL–6 plays an important role in the proliferation and differentiation of cells in humans32. IL–6 signalling relies on three distinct modes by the binding to three different targets: the transmembrane IL–6 receptor (mIL–6R, classic mode), the soluble forms of IL–6R (sIL–6R, trans-signaling mode), and the signal-transducing subunit molecule gp130 (trans-presentation mode, joined through IL–6R to gp130 on nearby located cells). These pathways, and the fact that gp130 is ubiquitously expressed, lead to the pleiotropic functions of IL–6 involved in diverse physiological processes and in the pathogenesis of different pathological conditions32 including the cytokine storm which has been identified as one of the major determinants of COVID–19 interstitial pneumonia. On the basis of today’s Italian experience (yet unpublished observations) COVID–19 pneumonia is somehow atypical as compared to the classic interstitial pneumonia: as compared to these latter recovers in longer period, thus posing a perduring threat to patient’s life which more frequently develop multi-organ failure. This is probably due to a persisting and overwhelming inflammation i.e - intriguingly - a condition where HDAC activity is involved.
A list of compound to test for COVID–19 infection has also been investigated by a computational model. Spike protein (S-protein) of SARS-COV–2 interacting with the human ACE2 receptor has also been used for docking-based screening to identify small molecules which bind to either the isolated Viral S-protein at its host receptor region or to the S protein-human ACE2 interface. The hypothesis used in this research considered that small molecules might limit viral recognition of host cells and/or disrupt host-virus interactions. We propose that the compounds emerging from the above analysis could be tested experimentally33. Between them, we considered the ones able to modulate HDAC activity (Table 3).
Table 3: Proposed compounds binding S-protein: ACE2 that modulate HDAC activity. Top ligands are based on Vina Score. In bold are indicated natural products.
Name (SWEETLEAD)
|
Vina Score
|
Benserazide (30)
|
- 7.400
|
Luteolin-monoarabinoside
|
- 7.400
|
Quercetol; quercitin
|
- 7.300
|
Protirelin (31)
|
- 7.300
|
Benserazide (30)
|
- 7.200
|
Vidarabine (32–40)
|
- 7.100
|
Some of them are natural products as luteolin and quercetin. Luteolin has been studied as an alternative therapy for hypercholesterolemia and associated cardiovascular diseases 34. Luteolin also showed a strong HDAC inhibitor activity and has been proposed for lung cancer therapies in together with other standard anticancer drugs (e.g., cisplatin) 35.
Quercetin has been identified as a SIRT6 inhibitor, modulating chromatin structure and showing a histone deacetylation inhibition higher than 50% 36.
A mixture of three different drugs has been suggested based on the activity they have by targeting HCoV-host network: sirolimus/dactinomycin; mercaptopurine/melatonin; and toremifene/emodin. They are natural compounds that may be considered as additive treatment and good candidates for COVID–19 treatment 37.
HDAC inhibitors as promising therapeutics for COVID–19 infection
A list of some EMA/FDA approved inhibitors to be considered for COVID–19 already used to cure other diseases is reported in Table 4. Other HDAC inhibitors have been considered in the clinical trials available on the ClinicalTrials.gov web site. (Table 5)
Table 4: EMA/FDA approved HDAC inhibitors.
Classification
|
Drug
|
HDAC specificity
|
Biologic event
|
Trial stage
|
|
|
|
|
|
Aliphatic fatty
|
Valproic acid
|
Classes I, II a
|
Class-switch DNA
|
Phase I and II trial
|
Acids
|
|
|
recombination (CSR)
|
|
|
|
|
|
|
|
|
and plasma cell
|
|
|
|
|
differentiation;
|
FDA Approved
|
|
|
|
|
for epilepsy,
|
|
|
|
CD20 expression
|
bipolar disorder
|
|
|
|
|
and migraine
|
|
|
|
|
|
Hydroxamate
|
SAHA (Vorinostat)
|
Classes I, II, and IV
|
CD20 expression;
|
FDA approved for
|
|
|
|
enhances apoptosis
|
cutaneous T cell
|
|
|
|
mediated by kinase
|
lymphoma (2006)
|
|
|
|
|
|
|
|
inhibitors that affect
|
|
|
|
|
|
|
|
BCR signaling and gene
|
|
|
|
|
expression disruption in
|
|
|
|
|
mantle cell lymphoma
|
|
|
|
|
|
|
|
PXD101(Belinostat)
|
Pan iHDAC
|
Cell-cycle arrest or
|
EMA approved
|
|
|
|
apoptosis of cancerous
|
for the treatment
|
|
|
|
cells
|
of patients with
|
|
|
|
relapsed or
|
|
|
|
|
|
|
|
|
refractory
|
|
|
|
|
peripheral T-cell
|
|
|
|
|
lymphoma (2014)
|
|
|
|
|
|
|
|
|
|
|
|
LBH589
|
Classes I, II, and IV
|
Reduced cell number
|
EMA approved
|
|
(Panobinostat)
|
|
and viability; delayed
|
for use and FDA
|
|
|
|
division progression;
|
accelerated
|
|
|
|
approval for use
|
|
|
|
decreases the number of
|
|
|
|
in multiple
|
|
|
|
CD138+ antibody-
|
|
|
|
myeloma (in
|
|
|
|
secreting cells
|
|
|
|
combination with
|
|
|
|
|
|
|
|
|
bortezomib and
|
|
|
|
|
dexamethasone)
|
|
|
|
|
(2015)
|
|
|
|
|
|
Cyclic peptides
|
FK228 (Romidepsin)
|
HDACs 1, 2
|
Reduced cell number
|
FDA approved for
|
|
|
|
and viability
|
cutaneous T cell
|
|
|
|
|
lymphoma (2009)
|
|
|
|
|
and for peripheral
|
|
|
|
|
T cell lymphoma
|
|
|
|
|
(2011)
|
|
|
|
|
|
Table 5: HDAC inhibitors (still on trial)
Classification
|
Drug
|
HDAC specificity
|
Biologic event
|
Trial stage
|
Aliphatic fatty acids
|
Butirate
|
Classes I, II a
|
Apoptosis, cell growth inhibition, cell cycle arrest, and cell differentiation
|
Phase I and II trial
Phase II and III trials, schizophrenia (NCT02654405; NCT03010865)*
|
|
AN-9 (Pivanex)
|
Classes I, II
|
Apoptosis, differentiation and reduced BCR-ABL protein levels
|
Phase II trial
|
Hydroxamate
|
ITF2357 (Givinostat)
|
Classes I, II
|
Cell proliferation inhibition and apoptosis induction in chronic myelogenous leukemia, BCR-ABL1-positive and childhood B acute lymphoblastic leukemia
|
Phase II trial
Duchenne muscular dystrophy
|
|
4SC201 (Resminostat)
|
Pan iHDAC
|
Inhibits proliferation of a large variety of rodent and human cancer cell lines
|
Phase II trial; advanced stage mycosis fungoides (NCT02953301)*
Phase II trial for relapsed or refractory Hodgkin's Lymphoma
(NCT01037478)*
Phase 2 hepatocellular carcinoma (NCT00943449)*
|
|
PCI24781(Abexinostat)
|
Classes I, II
|
Induces caspase and reactive oxygen species-dependent apoptosis through NF-kappa B mechanisms
|
Phase I- II trials for B-cell lymphom;
(NCT01027910)*
|
|
LAQ-824 (Dacinostat)
|
Pan iHDAC
|
Decreases viability in B-ALL, multiple myeloma, and B lymphoma cells
|
No trials Registered
|
Table 5 (continued): HDAC inhibitors (still on trials).
Hydroxamate
|
TSA (Trichostatin A)
|
Class I and II
|
CD20 expression (Raji cells)
Dose-dependent proliferation inhibition (CLBL-1 cells)
|
No trials Registered
|
|
ACY-241 (Cytarinostat)
|
HDACs 1, 2, 3, and 6
|
Inhibition of plainhisma cell myeloma proliferation and survival; cell cycle disruption
|
Phase I trial; multiple myeloma (NCT02886065)*
|
|
ACY-738
|
HDACs 1, 2, 3, and 6
|
Pre-B cell growth inhibition in lupus disease
|
No trials Registered
|
|
Acy-1215 (Riconilostat)
|
HDAC 6
|
CD20 expression
|
Phase II trial; diabetic neuropathic pain (NCT03176472)*
|
|
Tubacin
|
HDAC 6
|
CD20 expression; dose-dependent proliferation inhibition
|
No trials Registered
|
|
BML-281
|
HDAC 6
|
Blocks B cell infiltration in acute colitis
|
No trials Registered
|
|
LMK-235
|
HDACs 4, 5
|
Induces apoptosis and BCLA1 overexpression in diffuse large B cell lymphoma
|
No trials Registered
|
|
RGFP966
|
HDAC 3
|
Induces apoptosis, decreases Bcl-2 and Bcl-xL expression. Myc-mediated miR expression
|
No trials Registered
|
Table 5 (continued): HDAC inhibitors (still on trials).
Benzamides
|
MS-275 (Entinostat)
|
HDACs 1, 3, 4, 6, 8, and 10
|
Proliferation inhibition and apoptosis induction; decreases cell viability in B-ALL, B-lymphoma, and multiple myeloma cell lines
|
Phase I and II -Hodgkin’s lymphoma
Phase III trial – Metastatic lung cancer
It has been approved in combination with anticancer tumor compounds
|
|
MGCD0103 (Mocetinostat)
|
Class I and IV
|
Modulation of immune response
|
Phase II trial, urothelial carcinoma (NCT02236195)*, metastatic leiomyosarcoma (NCT02303262)*,
non-small cell lung cancer (NCT02954991)*
|
|
CI- 994 (Tacedinaline)
|
Class I
|
Proliferation inhibition and apoptosis induction
|
Phase II and III trials, lung cancer (NCT00005093)*
|
|
AR-42
|
Class I and II
|
Cell-cycle arrest and apoptosis via both caspase-3-dependent and caspase-3-independent pathways
|
Phase I trial; renal cell carcinoma or soft tissue sarcoma (NCT02795819)*, acute myeloid leukemia (NCT01798901)*, multiple myeloma (NCT02569320)*
|
Cyclic peptides
|
Apicidin
|
Class I
|
Cell growth and cell proliferation inhibition
|
No trials Registered
|
Mercaptoketone
|
KD5170
|
1 and 2
|
Apoptosis
|
No trials Registered
|
Sirtuins inhibitors
|
Nicotinamide
|
all Class III
|
Cell cycle arrest and autophagy
|
Phase III trial
|
|
Sirtinol
|
SIRT 1 and 2
|
Apoptosis and autophagic cell death
|
Preclinical
|
|
Cambinol
|
SIRT 1 and 2
|
Inhibits SIRT1 and 2 by induced hyperacetylation of p53
|
Preclinical
|
|
EX-527
|
SIRT 1 and 2
|
Inhibits SIRT1 by induced hyperacetylation of p53
|
Preclinical; phase I and II trials
|