key: cord-0948521-67d8o7o4
authors: Du, Ashuai; Zheng, Rong; Disoma, Cyrollah; Li, Shiqin; Chen, Zongpeng; Li, Sijia; Liu, Pinjia; Zhou, Yuzheng; Shen, Yilun; Liu, Sixu; Zhang, Yongxing; Dong, Zijun; Yang, Qinglong; Alsaadawe, Moyed; Razzaq, Aroona; Peng, Yuyang; Chen, Xuan; Hu, Liqiang; Peng, Jian; Zhang, Qianjun; Jiang, Taijiao; Mo, Long; Li, Shanni; Xia, Zanxian
title: Epigallocatechin-3-gallate, an active ingredient of Traditional Chinese Medicines, inhibits the 3CLpro activity of SARS-CoV-2
date: 2021-02-04
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2021.02.012
sha: 3a2d09f34abe1184d219112ea67bac593ef0bd24
doc_id: 948521
cord_uid: 67d8o7o4

SARS-CoV-2 is the etiological agent responsible for the ongoing pandemic of coronavirus disease 2019 (COVID-19). The main protease of SARS-CoV-2, 3CLpro, is an attractive target for antiviral inhibitors due to its indispensable role in viral replication and gene expression of viral proteins. The search of compounds that can effectively inhibit the crucial activity of 3CLpro, which results to interference of the virus life cycle, is now widely pursued. Here, we report that epigallocatechin-3-gallate (EGCG), an active ingredient of Chinese herbal medicine (CHM), is a potent inhibitor of 3CLpro with half-maximum inhibitory concentration (IC50) of 0.874 ± 0.005 μM. In the study, we retrospectively analyzed the clinical data of 123 cases of COVID-19 patients, and found three effective Traditional Chinese Medicines (TCM) prescriptions. Multiple strategies were performed to screen potent inhibitors of SARS-CoV-2 3CLpro from the active ingredients of TCMs, including network pharmacology, molecular docking, surface plasmon resonance (SPR) binding assay and fluorescence resonance energy transfer (FRET)-based inhibition assay. The SPR assay showed good interaction between EGCG and 3CLpro with KD ~6.17 μM, suggesting a relatively high affinity of EGCG with SARS-CoV-2 3CLpro. Our results provide critical insights into the mechanism of action of EGCG as a potential therapeutic agent against COVID-19.

The SARS-CoV-2 is the seventh coronavirus that is able to infect humans with contagious and infective characteristics [5] . According to World Health Organization (WHO), the virus has infected thus far more than 53 million people worldwide, resulting the death of 1,308,975 people as of 15 November 2020 [6] . Effective pharmacological interventions are therefore urgently needed. Since the first report of COVID-19 in late 2019, relevant progress has been achieved in drug discovery and development within a remarkable timeline [7] [8] [9] [10] [11] . Several vaccines and drugs are undergoing clinical trials, but research on novel antivirals remains important. This is because of the growing clinical needs and also of the possible viral mutations that may render previous treatments ineffective.

In terms of drug discovery approaches against coronaviruses, the main protease (Mpro) has known with a similar cleavage specificity, targeting 3CLpro offers the advantage of virusspecific effect [14, 15] . Moreover, 3CLpro is highly conserved in coronaviruses (CoVs). In fact, the sequence similarity between SARS-CoV and SARS-CoV-2 is as high as 96.08% [15, 16] .

Acting as a broad antiviral treatment option, drugs targeting 3CLpro can be crucial not only to inhibit SARS-CoV-2 but as well as other future coronavirus variants. For these reasons, 3CLpro has been well studied particularly following the SARS-CoV outbreak in 2002 [12, 17, 18] . The past experience with SARS-CoV is providing important foundational knowledge for the development of anti-SARS-CoV-2 drugs.

There are several ways to obtain small molecules that could be developed as viral target protein inhibitors. The first major option is in silico screening of large chemical libraries to select only the molecules that best fit with the target of interest for synthesis and experimental testing [19] . Other option is through structure-based drug design and chemical synthesis, such as the broad-spectrum inhibitor N3 that inhibits 3CLpro of SARS-CoV, MERS-CoV and SARS-CoV-2 [14, 20, 21] . An additional approach is bioprospecting in which potent compounds are identified and isolated from natural sources e.g. plant varieties. Over the past few decades, natural compounds of plant-based origin have been extensively studied as an exciting class of pharmacologically active molecules [22] [23] [24] . In particular, many natural products have demonstrated potent activity against CoVs [25] . Glycyrrhizic acid, baicalin, quercetin have been reported to inhibit the replication of SARS-CoV in vitro [26] [27] [28] . Also, natural products including betulinic acid, indigo, flavone amentoflavone, and luteolin have also been identified to inhibit the enzymatic activity of SARS-CoV 3CLpro [29, 30] . Hence, the screening of inhibitors for SARS-CoV-2 3CLpro from natural products is a worthwhile direction.

Chinese herbs are great sources of natural compounds. Many drugs that are used in the clinics were derived from Chinese herbs. Traditional Chinese Medicine has played an important role in the treatment of the past epidemics caused by viral infections. Notably, it has received renewed attention during the outbreak of COVID-19. Following reports of good clinical efficacy, several TCMs were officially endorsed for clinical use in China and were adopted as part of the treatment plan for COVID-19 [31] [32] [33] . Increasing number of clinical and experimental evidences have proved that TCMs inhibit viral replication owing to specific active ingredients [34] [35] [36] .

Herein, we retrospectively analysed 123 COVID-19 patients who received a combination of western and Traditional Chinese Medicines from the hospitals in Guizhou Province, China.

Three TCM prescriptions were found to be effective. Multiple computational and experimental compounds can be used as drugs. In this study, OB ≥ 30% and DL ≥ 0.18 were used as screening threshold. Then, the targets of such compounds were obtained from the TCMSP database, in which the components not included in TCMSP were predicted through The Encyclopedia of Traditional Chinese Medicine database (ETCM; http://www.tcmip.cn/ETCM/index.php/Home/). Finally, the target proteins were identified using UniProt Database (https://www.uniprot.org/).

The associated genes to COVID-19 were searched in GeneCard database (https://www.genecards.org) and NCBI (https://www.ncbi.nlm.nih.gov/) using the keyword "coronavirus pneumonia".

The seven most frequent compounds common among the three CHM formulas were docked with the key target SARS-CoV-2 3CLpro hydrolase. Chem3D software was used to draw the 3D structures of the core compounds and to optimize the energy. The protein crystal structure of 3CLpro CoV-2 3CLpro recombinant protein (Novoprotein #CR76) was over 95%.

The thermal shift assay (TSA) was performed using CFX96 Touch Real-Time PCR Systems The initial thermal denaturation temperature was set at 25°C for 2min. The temperature was then systematically increased in 0.5-1.0 °C up to final temperature of 95 °C, with concomitant monitoring of fluorescence emission at the end of every 1 min hold at each temperature. Two methods were used to calculate the melting temperature (T m ) as previously described [37] . In brief, one method was used to determine the melting temperature from nonlinear fitting of the thermal denaturation data. The other method involved calculating the first derivative of fluorescence emission with respect to temperature. Since thermal stability of 3CLpro with the addition of quercetin was recently reported [16] , quercetin was used as a positive control to test the feasibility of the PCR system. To assess the concentration dependence of the stability change induced by EGCG, 2-fold serial dilutions ranging from 0 to 250 M were assayed by following the same protocol described above.

A fluorescence resonance energy transfer (FRET) protease assay was applied to measure the inhibitory activity of compounds against the SARS-CoV-2 3CLpro. The fluorogenic substrate 

Continuous variables were expressed as mean, median and interquartile (IQR). Categorical data were expressed as number (%). Categorical variables were compared by Chi-square (χ2) test.

Line graphs were drawn to describe laboratory parameters. The distribution of normality was checked by Kolmogorov-Smirnov test. When data were not normally distributed, Mann-Whitney test was used to determine statistical significance. A p value of <0.05 was considered significant.

Statistical analyses were performed using the SPSS software, version 23.

Out P<0.05 was considered significant. COPD, chronic obstructive pulmonary disease; ARDS acute respiratory distress syndrome. One patient died of ARDS.

Laboratory values and radiographic findings at the time of admission are presented in Table 2 .

Regardless of the disease severity, all 123 patients had white blood cell, neutrophil, lymphocyte, and platelet counts within the normal ranges. At the time of admission, the median values for Creactive protein, erythrocyte sedimentation rate, procalcitonin, creatine kinase, creatine kinase isoenzyme, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, and lactate dehydrogenase were also all within the normal ranges. There was no statistically Data are presented as median (IQR) or n (%). P values indicate difference among groups (mild, moderate and severe). P < 0.05 was considered significant. NA, not available.

Drug treatment included commercially available drugs and TCMs including antivirals, antibiotics, antihypertensive drugs, lipid-lowering drugs, digestive system drugs, respiratory drugs, anti-hypoglycemics, antipyretics, analgesics and others. Antihypertensive drugs, lipidlowering drugs, hypoglycemic drugs, and gastrointestinal medications were mainly used for the treatment of patients with underlying diseases, whereas respiratory drugs, antipyretics and analgesics were used for symptomatic treatment. Of the 20 severe patients, 4 (3.3%) were admitted to the ICU and all received high-flow oxygen.

Among them, one patient received non-invasive ventilation while others were treated with convalescent plasma therapy. There was no statistically significant difference in the application of other drugs among groups as well as the use of Chinese medicine. 

To determine the clinical features associated with COVID-19 progression, six clinical laboratory parameters were tracked in 60 patients every 2 days for a period of two weeks from the onset of symptoms ( Figure 2 ). Biochemical indicators (white blood cell, neutrophil, erythrocyte sedimentation rate, C-reactive protein, and D-dimer) in severe patients were higher than those of mild and moderate patients. But at the end of hospital admission, these biochemical parameters in severe patients decreased. After 1 week of hospitalization, all these laboratory findings in all our patient types subsided, which was related to faster recovery and shorter hospital stay. Taken the clinical data altogether, the use of TCMs seemed effective regardless of disease severity and appeared to modulate immune responses.

Our patient cohort received 9 different TCM prescriptions in total. Among these, three prescriptions, namely, Yangyinjiedu (YYJD), Dayuanxiaodu (DYXD) and Chaihuqingzao (CHQZ) were selected for further evaluation because they were used most frequently. A network pharmacology method was conducted to investigate their effectiveness for COVID-19. We These pathways included TNF signaling, IL-17 signaling, influenza A, C-type lectin receptor signaling pathway, and others (Supplementary Figure S3) . These GO, KEGG as well as the clinical data all showed the immunomodulatory effects of these three TCMs.

The active compounds of the three TCM prescriptions were retrieved from the TCMSP and ETCM databases (Supplementary Tables S1, S2 and S3). Some active ingredients are common among all the three TCMs. Thereafter, we further determined the frequencies of these ingredients and noted their curated mechanism of actions. This screening strategy resulted to 7 compounds that are possibly responsible for the anti-COVID-19 properties of the TCMs. These compounds are quercetin, kaempferol, luteolin, isorhamnetin, epigallocatechin-3-gallate, naringenin, and wogonin. These compounds may underpin the antiviral activity of these TCMs, but the exact mechanism is unknown. While every viral enzyme of SARS-CoV-2 is a potential drug target, 3CLpro was selected to be the focus of our further analyses because of its indispensable function in the viral life cycle. The replication of SARS-CoV-2 is mediated by replicase polyprotein, consisting of pp1a and pp1ab. This polyprotein is then cleaved to form 16 functional polypeptides, also called non-structural protein, via extensive proteolytic processing predominantly by 3CLpro [38] . Thus, 3CLpro plays a critical function in the life cycle of SARS-CoV-2. More so, the cleavage sites of 3CLpro are highly conserved in CoVs with no known human proteases having similar cleavage specificity, providing added advantage of possible less toxicity of 3CLpro inhibitors [15] . With these reasons-functional importance in viral life cycle, high similarity of 3CLpro among CoVs, and cleavage specificity-targeting 3CLpro of SARS-CoV-2 is an attractive antiviral strategy. We therefore docked the compounds with 3CLpro using AutoDock Vina1.1.2. It is generally believed that the lower energy of the conformational stability of the ligand and the receptor, the greater the possibility of interaction. When the binding energy ≤-5.0 kcal /mol is taken as the screening criterion, the results of molecular docking showed that all the compounds were all lower than -5 kcal/mol (Table 4 ). This indicates that they had a certain affinity for the protein crystal structure of 3CLpro ( Figure 3) Figure 4E ). Besides these two key residues, several other amino acid residues in the active site were also involved in hydrogen bonding as well as different noncovalent interactions. Similarly, quercetin is also able to form various interactions with a number of residues of the binding pocket [16] . The key residue is Met165, which can form both hydrogen bonds and hydrophobic interactions with quercetin.

Additional hydrogen bonds can be formed with the two residues Ser144 and Met165. Energetic contributions are also due to supplementary hydrophobic interactions with Met49, Phe140 and Leu141, and to electrostatic interaction formed with the polar residue His164 and the charged residue Glu166. All in all, EGCG and quercetin share certain similarity in terms of binding affinity with some key residues in the active site of 3CLpro. But since EGCG appears to interact with the catalytic residues of the 3CLpro protease and exhibited a more favorable binding affinity, it is likely that EGCG possibly inhibits the catalytic activity of 3CLpro. This therefore renders EGCG as a good candidate for drug development for the treatment of COVID-19. and EC50 of 4.670.80M [27] . Though wogonin and isorhamnetin were also predicted to have favorable affinity with 3CLpro, they were not analyzed by SPR due to its low water solubility.

The in silico docking study and SPR binding assay both indicated that the active compounds have certain affinity with 3CLpro, the key protease during SARS-CoV-2 replication. The anti-COVID-19 property of these compounds is possibly due to its ability to inhibit the enzymatic activity of 3CLpro. In this regard, a fluorescence resonance energy transfer (FRET)-based cleavage assay was used to determine the median inhibitory concentration (IC50) values. As 

Generally, the stability of a protein can be affected by the interaction with a ligand [39] . Because of its easy use, thermal shift assay (TSA) has become an excellent platform for discovery of small molecule ligands [37] . To further characterize the potentials of EGCG as 3CLpro inhibitor,

we performed TSA to determine if such compound alters thermal stability of 3CLpro. Since quercetin alters the thermal stability of 3CLpro by causing destabilization [16] , we used quercetin as our positive control. To validate our own experimental system, we performed TSA on quercetin with 3CLpro. Compared with the control group (DMSO only), the addition of quercetin reduced the melting temperature (T m ) of 3CLpro ( Figure 6A ), consistent with the previous report. This indicates that the experimental system is suitable. Similar to quercetin, EGCG also altered the thermal stability of SARS-CoV-2 3CLpro in a dose-dependent manner ( Figure 6B ) with a melting temperature (T m ) of 53.80C at a concentration of 62.5M ( Figure   6D ).

The lack of mass immunization programs and antiviral drugs continues to be the serious challenge to the current global effort to contain the COVID-19 epidemic. Several drugs were tested in randomized controlled clinical studies but unfortunately the vast majority of these drug interventions were not successful [40] [41] [42] . Therefore, different drug discovery strategies need to be considered in addition to the current widely used drug repurposing methods and structurebased drug design strategies. In this study, several natural compounds from Traditional Chinese

Medicine were identified as SARS-CoV-2 3CLpro inhibitors. Here, we retrospectively evaluated medical records of patients who received a combination of Chinese and Western medicines.

Three effective TCMs were identified, namely Yangyinjiedu, Dayuanxiaodu and Chaihuqingzao.

Moreover, the active components of these three herbs were identified by network pharmacology.

Additionally, the active compounds with potential therapeutic value for COVID-19 were screened through in vitro experiments.

In [43] [44] [45] [46] [47] . In addition, TCM is widely used in clinical practice in the treatment of viral infectious diseases. For example, Lian-Hua-Qing-Wen Capsule (LHQWC), a commonly used Chinese medicine, was approved for the treatment of SARS and viral influenza [48, 49] . In the height of SARS-CoV-2 outbreak in China, LHQWC was recommended again for clinical use to treat COVID-19. Subsequent clinical and experimental studies have confirmed the therapeutic benefits of LHWQC [35, 50] . Its mode of action was by targeting virus replication and immunological regulation. In addition to LHQWC, several other TCMs have also been shown to be effective against COVID-19 and have been recommended for clinical use [51, 52] . Therefore, it may be an economical and time-saving pursuit to screen inhibitors from effective TCM formulations.

Due to the critical function of 3CLpro in the life cycle of SARS-CoV-2, we screened several active ingredients against 3CLpro from TCMs. Our molecular docking analysis revealed that seven core compounds from the three effective TCMs are predicted to have good binding affinity with 3CLpro. Our SPR binding assay further confirmed the predicted affinity of the compounds with 3CLpro. This potential binding may directly inhibit its proteolytic activity. To test this hypothesis, we evaluated if the compounds can inhibit the enzymatic activity of 3CLpro using FRET assay. Of the soluble compounds, EGCG has the best inhibitory effect with IC50 of 0.874±0.005μM.

EGCG is a polyphenol catechin that is abundant in tea plants, especially green tea [53] .

EGCG showed a wide range of antiviral activity against adenovirus, influenza virus, zika virus, herpesvirus, and hepatitis virus [54] . It has also been found to be a potential treatment option over synthetic chemical drugs. In addition, previous studies have reported that EGCG also has antiviral activity against coronavirus. In vitro studies showed that EGCG inhibited SARS-CoV 3CLpro with an IC50 value of 73±2 μM [29] . Given the high similarity of 3CLpro in SARS-CoV and SARS-CoV-2, EGCG is a potential inhibitor of 3CLpro of SARS-CoV-2 and can be a lead candidate for drug development to treat COVID-19. Our molecular docking results showed that EGCG, with a docking score of −7.9, has the best in silico activity among all the compounds tested. This is consistent with the results of several recent reports [55, 56] [58, 59] . Moreover, EGCG can suppress the canonical NF-κB pathway [60, 61] , which plays a key regulatory role in the expression of proinflammatory cytokines including IL-1, TNF-α, IL8, IL-6. All of these cytokines are induced in cytokine storm syndrome as well as in COVID-19. The KEGG analysis and the GO-based enrichment analyses also showed that a vast majority of the most enriched pathways of the components in the three TCMs were associated with immune response and inflammation-related signalling (Supplementary Figure S2A , S2B, S2C and S3). As illustrated in the compound-target regulation network diagram ( Figure S1 ), several active components interact with a variety of targets. In particular, EGCG (ID：MOL006821) is predicted to target multiple genes. This prediction model provides a good insight but more research efforts are still needed to fully uncover the therapeutic benefits of EGCG.

In this work, we screened and identified several active ingredients of Traditional Chinese

Medicine with inhibitory activity against SARS-CoV-2 3CLpro. Among them, EGCG is the most promising active compound due to its inhibitory activity. EGCG offers additional J o u r n a l P r e -p r o o f

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

of Novel Coronavirus-Infected Pneumonia

World Health Organization Coronavirus Disease

COVID-19 update: The race to therapeutic development

Accelerated Preclinical Paths to Support Rapid Development of COVID-19 Therapeutics

Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2

Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs

From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design

Design of wide-spectrum inhibitors targeting coronavirus main proteases

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening

An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

Activation and maturation of SARS-CoV main protease

Ultra-large library docking for discovering new chemotypes

Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design

The newly emerged SARS-like coronavirus HCoV-EMC also has an "Achilles' heel": current effective inhibitor targeting a 3C-like protease

Nature-derived compounds modulating Wnt/ beta -catenin pathway: a preventive and therapeutic opportunity in neoplastic diseases

Roles of plant-derived bioactive compounds and related microRNAs in cancer therapy

Bioactive natural compounds against human coronaviruses: a review and perspective

Current Prevention of COVID-19: Natural Products and Herbal Medicine

Antiviral activity of glycyrrhizic acid derivatives against SARS-coronavirus

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features

Roles of flavonoids against coronavirus infection

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition

Profiles of COVID-19 clinical trials in the Chinese Clinical Trial Registry

China National Health Comission Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and

Becoming a Faithful Defender: Traditional Chinese Medicine against Coronavirus Disease 2019 (COVID-19)

Shufeng Jiedu, a promising herbal therapy for moderate COVID-19:Antiviral and anti-inflammatory properties, pathways of bioactive compounds, and a clinical real-world pragmatic study

Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2)

Identifying potential anti-COVID-19 pharmacological components of traditional Chinese medicine Lianhuaqingwen capsule based on human exposure and ACE2 biochromatography screening

Analysis of protein stability and ligand interactions by thermal shift assay

Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors

Looking for protein stabilizing drugs with thermal shift assay

A pragmatic randomized controlled trial reports lack of efficacy of hydroxychloroquine on coronavirus disease 2019 viral kinetics

Antiviral traditional medicines against herpes simplex virus (HSV-1), poliovirus, and measles virus in vitro and their therapeutic efficacies for HSV-1 infection in mice

Antiviral potential of natural compounds against influenza virus hemagglutinin

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Effective inhibition of MERS-CoV infection by resveratrol

Antiviral potential of medicinal plants against HIV, HSV, influenza, hepatitis, and coxsackievirus: A systematic review

Effect of lianhuaqingwen capsules on airway inflammation in patients with acute exacerbation of chronic obstructive pulmonary disease

The Chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function

Efficacy of Early Combination Therapy With Lianhuaqingwen and Arbidol in Moderate and Severe COVID-19 Patients: A Retrospective Cohort Study

Chemical composition and pharmacological mechanism of Qingfei Paidu Decoction and Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In silico and experimental study

The clinical benefits of Chinese patent medicines against COVID-19 based on current evidence

Recent Advances in the Understanding of the Health Benefits and Molecular Mechanisms Associated with Green Tea Polyphenols

Tea Polyphenols EGCG and Theaflavin Inhibit the Activity of SARS-CoV-2 3CL-Protease In Vitro

Identification of bioactive molecules from tea plant as SARS-CoV-2 main protease inhibitors

Molecular docking and dynamic simulations for antiviral compounds against SARS-CoV-2: A computational study

Cytokine elevation in severe and critical COVID-19: a rapid systematic review, meta-analysis, and comparison with other inflammatory syndromes

Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

Protective Effect of Epigallocatechin-3-Gallate (EGCG) in Diseases with Uncontrolled Immune Activation: Could Such a Scenario Be Helpful to Counteract COVID-19?

Protective effect of green tea catechin against urban fine dust particle-induced skin aging by regulation of NF-kappaB, AP-1, and MAPKs signaling pathways

The tea catechin epigallocatechin gallate inhibits NF-kappaB-mediated transcriptional activation by covalent modification

Potential Therapeutic Targets of Epigallocatechin Gallate (EGCG), the Most Abundant Catechin in Green Tea, and Its Role in the Therapy of Various Types of Cancer