key: cord-0882423-u4ip47ll
authors: Gubarev, Yury A.; Lebedeva, Natalya Sh; Yurina, Elena S.; Syrbu, Sergey S.; Kiselev, Aleksey N.; Lebedev, Mikhail A.
title: Possible therapeutic targets and promising drugs based on unsymmetrical hetaryl-substituted porphyrins to combat SARS-CoV-2
date: 2021-08-05
journal: J Pharm Anal
DOI: 10.1016/j.jpha.2021.08.003
sha: 3156124c6e7ec9262b1c8b8cd60954bc4e7a98cb
doc_id: 882423
cord_uid: u4ip47ll

Coronavirus disease 2019 is a serious disease that causes acute respiratory syndrome and negatively affects the central nervous system. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) crosses the blood-brain barrier due to the S protein on the surface of the viral particles. Thus, it is important to develop compounds that not only have an inhibitory effect but are also capable of completely deactivating the S-protein function. This study describes the purposeful modification of porphyrins and proposes compounds, asymmetrically hetaryl-substituted porphyrins with benzothiazole, benzoxazole, and N-methylbenzimidazole residues, to deactivate the S-protein functions. Molecular docking of SARS-CoV-2 proteins with hetaryl-substituted porphyrins showed that the viral proteins S-, N-, and nsp13 exhibited the highest binding affinity. Hetaryl-substituted porphyrins form strong complexes (13–14 kcal/mol) with the receptor-binding domain of the S protein, while the distance from the porphyrins to the receptor-binding motif (RBM) does not exceed 20 Å; therefore, RBM can be oxidized by (1)O(2), which is generated by porphyrin. Hetaryl-substituted porphyrins interact with the nucleocapsid protein in the serine/arginine-rich region, and a number of vulnerable amino acid residues are located in the photooxidation zone. This damage complicates the packaging of viral RNA into new virions. High-energy binding of hetaryl-substituted porphyrins with the N- and C-terminal domains of nsp13 was observed. This binding blocks the action of nsp13 as an enzyme of exoribonuclease and methyltransferase, thereby preventing RNA replication and processing. A procedure for the synthesis of hetaryl-substituted porphyrins was developed, new compounds were obtained, their structures were identified, and their photocatalytic properties were studied.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory 32 syndrome coronavirus 2 (SARS-CoV-2) has clearly demonstrated the vulnerability and 33

defenselessness of humanity to new viral threats. COVID-19 usually causes severe acute 34 respiratory syndrome [1] . In addition, new clinical reports are increasing, indicating the dramatic 35 effects of SARS-CoV-2 on the central nervous system. For example, a previous study [2] reported 36 that SARS-CoV-2 preferentially targeted neurons in the brain. Imaging of neurons shows that 37 exposure to SARS-CoV-2 is associated with altered tau protein distribution and 38 hyperphosphorylation, ultimately resulting in neuronal death. 39

The neurological symptoms of COVID-19 are complex and differ among individuals [3] . 40 Considering this symptomatology, two possible mutually non-exclusive forms of virus penetration 41

were proposed: through the olfactory nerves (transneuronal) and the hematogenous pathway with 42 penetration via the blood-brain barrier (BBB). In a cytokine storm, BBB permeability is impaired. 43 Several attempts have been made to determine the exact cause of damage to the central nervous 44 system, such as direct penetration of the virus into the brain by binding to the BBB cells or 45 penetration of the virus due to increased BBB permeability during a cytokine storm. To date, the 46 confirmed cause of central nervous system damage is the direct penetration of virus. As a result, 47

the BBB becomes vulnerable due to the presence of S protein on the surface of the virus, which is 48 the driving unit. A previous study [4] showed that the S-protein SARS-CoV-2 easily crosses the 49 BBB of mice, penetrates into the parenchymal tissue of the brain, and binds to the glycocalyx of 50 the capillaries of the brain, causing damage and apoptosis of endothelial cells and neurons [5] . 51

Human neurodegenerative diseases are often a gradual process that, in some cases, develop 52 over several decades. Neurological and neurodegenerative disorders occur in approximately 35% 53 of patients [6, 7] . Given the possible long-term impact of COVID-19, it is necessary to develop 54 drugs that target the S-protein, not just to inhibit its binding to the angiotensin-converting enzyme 55 2 (ACE2), but to completely disrupt its functionality. 56

Recent studies showed that porphyrins and their structural analogues can be successfully used 57

to inactivate viruses and drug-resistant forms of pathogenic bacteria. Our study aimed to design 58 new chemical compounds with the desired properties, namely, hetaryl-substituted porphyrins, 59

using computer methods (molecular docking). In particular, the desired properties are as follows: 60 1) high-energy binding to the SARS-CoV-2 spike protein in the receptor-binding domain (RBD) 61 site or in its immediate vicinity (inhibition of binding to ACE2), 2) the ability to absorb light 62 energy in the far-wave part of the spectrum (for working in the "therapeutic window") and generate 63 reactive oxygen species (for irreversible damage to the protein during photoirradiation), 3) high 64 quantum yield of reactive oxygen species (for irreversible damage to the protein during 65 photoirradiation), 4) solubility in physiological media, and 5) the absence of specific interactions 66 (π-π, H-binding between the substance and amino acids of the S protein) to avoid additional 67 dissipation of light energy and the quantum yield of singlet oxygen increase when porphyrin binds 68

to the spike protein of the virus. 69

The D-I-TASSER-predicted structures of SARS-CoV-2 proteins were downloaded from the 72 Zhang Lab website [8, 9] . The heterotrimeric structure of Spike porphyrin (7 bnm) was downloaded 73 from the Protein Data Bank [10] . The structures of the porphyrins (Fig. 1) The residue in the flask was diluted with 300 mL of methanol and 30 mL of ammonia solution. 92

The precipitate of the porphyrin mixture was filtered, washed with methanol, and dried at room 93 temperature to achieve a constant weight. The dried precipitate was dissolved in 200 mL of 94 dichloromethane and chromatographed on a column with Al2O3 (activity grade III according to 95

Brockmann), eluting with a mixture of ethanol and methylene chloride (10:1, V/V). The third 96 fraction of 5-(4′-bromophenyl)-10,15,20-tris-(3′-pyridyl)porphyrin was collected. The solvent was 97 evaporated, and the porphyrin was rechromatographed on a column with Al2O3 (activity grade III 98 according to Brockmann), eluting with a mixture of ethanol and methylene chloride (10:1, V/V). 99

The product was monitored by thin-layer chromatography (TLC) on Alufol, ultraviolet-visible 100 (UV-Vis) spectrophotometry, 1 H NMR spectroscopy, and mass spectrometry (MALDI-TOF). Monitoring was carried out using a UV-VIS spectrometer. The mixture was cooled, and the excess 112 solvent was distilled, chromatographed on a column with Al2O3 (activity grade III according to 113

Brockmann), and eluted with a mixture of ethanol and methylene chloride (10:1, V/V). The solvent 114

was evaporated under vacuum, and the residue was washed with water, filtered, and dried at room 115 J o u r n a l P r e -p r o o f temperature to achieve a constant weight. Yield: 3.2 g (98% to propose structures of porphyrins that are believed to have the above specified properties (Fig.  215  1) . 216

To test our hypothesis using bioinformatics methods, hetaryl-substituted porphyrins were 217 docked with all SARS-CoV-2 proteins. The results are shown in Table 1 . Based on the data 218 obtained, hetaryl-substituted porphyrins exhibited the highest affinity for the S protein, followed 219 by the nucleocapsid protein and the nsp13 protein. The rest of the SARS-CoV-2 proteins are also 220 able to bind to hetaryl-substituted porphyrins, but with much less energy gain (Table 1) . 221

Let us consider the results obtained for the docking of hetaryl-substituted porphyrins with S 222 protein, N-proteins, and nsp13. The spike protein protruding on the surface of the viral particle 223

consists of three identical S protein chains. Each chain consists of two subunits, S1 and S2. The 224 S1 subunit is responsible for binding to receptors [21] . The S2 subunit is responsible for fusion of 225 the virus and host cell membranes. The S1 subunit includes a receptor-binding domain, an RBD 226 site that binds complementarily to the ACE2 receptor of the host cell. This interaction caused a 227 strong conformational change in the S2 subunit. Then, with the help of the host enzymes (furin 228 and transmembrane serine protease), the S protein is cleaved into the S1 and S2 subunits [22,23]. 229

Ultimately, it leads to the fusion of the viral envelope and the host cell membrane, followed by the 230 release of the nucleocapsid into the cytoplasm [24,25]. Thus, the S protein is an obvious target. 231

Targeting the S protein, especially in the RBD region, can inhibit viral entry into the cells. As an 232 example, Fig. 2 shows the result of hetaryl-substituted porphyrin docking containing a 233 benzoxazole residue with the S protein. The binding of all studied arylporphyrins occurs at the 234 same sites of the S protein, namely, on the RBD surface of each chain. More precisely, the 235 porphyrin compound is located between the RBD regions of two adjacent chains, simultaneously 236 interacting with both RBD amino acid sequences ( Phe486, and Ser494 bind to ACE2. We determined the distance between the amino acid residues 250 of RBM and the center of the hetaryl-substituted porphyrins (Table 3 ). 251

We believe that this is a very important characteristic, since the removal of the photosensitizer 252 from the indicated amino acid residues determines the possibility of complete blocking of the 253 binding of the virus to ACE2. The reactive oxygen species generated by photoirradiation of 254 porphyrin are very reactive. They have a very short lifetime and a short diffusion path. For singlet 255 oxygen, the estimated lifetime in water is 4 µs [28], and the maximum possible path of 1 O2 in the 256 absence of quenchers is no more than 100-150 nm [29] [30] [31] . This distance is much greater than the 257 distance of the RBM amino acid residues from the hetaryl-substituted porphyrins (Table 3) . 258

Therefore, upon photoirradiation of the complex, the probability of oxidation by the generated 259 porphyrins 1 O2 is extremely high. 260

The geometry of the complex and the nature of the interaction of S-protein with the 261 heteroatom had no influence on the composition of the peripheral porphyrin substituent (Table 2) . 262

The nature of the interaction of hetarylporphyrins with S-protein is universal and specific. For all 263 studied porphyrins,  binds to the Tyr369 amino acid residue of each S-protein. The arrangement 264 of the aromatic systems within the complexes was not strictly parallel (Fig. 3) . The average 265 interplanar distance was approximately 4 Å, which is typical for the - complexes of porphyrins. This value is too high for the H-bond; therefore, it  274 can be considered to be weak. This does not significantly affect the energy of the complex as a 275

whole. In addition to the S-protein, the studied hetaryl-substituted porphyrins demonstrated high 276 affinity for binding to the nucleocapsid protein and helicase (Table 1) atom of the peripheral porphyrin substituent ( Table 1 ). The SR region is important for condensing 290 the nucleocapsid protein with RNA through liquid-phase separation [34] . In the immediate amino 291 acid environment of hetarylporphyrins, the complex with the nucleocapsid protein contains amino 292 acid residues that are easily photooxidized. These include Tyr333, Trp301, Arg259, and Arg277. 293

The rate constant of their reaction with 1 O2 is higher than those of other amino acids and is (1-7) 294 × 10 7 M −1 ·sec −1 [35, 36] . We expect that photodamage to this area will have significant inhibitory 295 and virucidal effects. 296

The SARS-CoV replication/transcription complex consists of at least 16 non-structural 297

proteins. This complex includes replication enzymes and proteins that carry out post-298 transcriptional RNA modification (processing). The nsp13 non-structural protein of the 299 coronavirus is unique because it performs the functions of both exoribonuclease (ExoN) and 300

(guanine-N7)-methyltransferase (N7-MTase domain of nsp13 (ExoN) (Fig. 5) . Of all the hetaryl-substituted porphyrins studied, the hydrogen 309 bonds between the NH groups of the reaction center of the porphyrin and TRP131 form porphyrins, 310 which contain benzothiazole and N-methylbenzimidazole residues. Moreover, the lengths of the 311 H-bonds (Table 1) indicate the low energy of the complexes formed. This fact probably does not 312 significantly affect the decrease in the photocatalytic activity of the considered porphyrins. 313

Analysis of the amino acid environment closest to the porphyrins in a complex with the ExoN 314 domain revealed that only one amino acid residue (Met57) was highly susceptible to 315 photooxidation by singlet oxygen [35] . 316

The complexes of hetaryl-substituted porphyrins with the C-tail domain of nsp13 are formed 317 due to universal interactions; in the case of porphyrins containing benzothiazole and benzooxazole 318 residues, these complexes are additionally stabilized by the H-bond between the NH groups of the 319 porphyrin macroring and Val287. However, as in the case of complex (1), the energy is very low 320

( Table 1 ). The nearest amino acid environment of hetaryl-substituted porphyrins in complex (3) These results demonstrate that hetaryl-substituted porphyrins are able to efficiently bind to 324 parts of the nsp13 protein that performs an enzymatic function, especially in the C-tail domain. 325

The latter is responsible for the mechanism of RNA capping, which plays a vital role in the escape 326 of viral RNA from immune cells. Therefore, the breaking of binding to porphyrin, and even its 327 photoinduced oxidation of nsp13, leads to the degradation of viral RNA. Ultimately, this makes 328 the replication cycle impossible. 329

We developed a procedure for the synthesis of hetaryl-substituted porphyrins and examined 330 their photochemical properties. The general scheme for the synthesis of water-soluble hetaryl-331 substituted porphyrins is shown in Fig. 6 . 332

The obtained asymmetrically substituted porphyrins have a sufficiently high quantum yield 333 of singlet oxygen: OPOR, 0.61; NPOR, 0.83; and SPOR, 0.86. Based on the obtained quantum 334 yield values, the most promising compounds are NPOR and SPOR, but their ability to oxidize 335 RBM requires experimental verification. We plan to conduct in vitro studies to investigate the 336 different processes involved in the interaction between SARS-CoV-2 proteins and NPOR and 337 SPOR compounds, and to evaluate the possibility of RBM binding to ACE2 in the presence of 338 porphyrins in the dark and under photoirradiation. This will allow the assessment of the inhibitory 339 and photovirucidal activity of NPOR and SPOR. 340

Purposeful synthesis was carried out, and compounds 5- CoV-2 proteins with hetaryl-substituted porphyrins made it possible to reveal three promising 350 targets for inhibition and complete deactivation. These include the S-protein, nucleocapsid protein, 351 and nsp13. We believe this is a good strategy for targeting a potential drug to multiple targets, 352 especially given the frequent mutations of the virus and the three-tiered defense (at different stages π-π-interaction between the porphyrin macroring and the Tyr369 of the "B" chain (4 Å) H-bond between the N atom of the О-por hetaryl substituent and Gln409 of the "A" chain (3.3 Å) Table 3 . Distance between amino acid residues receptor-binding motif (RBM) and the center 5 of hetaryl-substituted porphyrins. 6

SARS-CoV-2 pathophysiology and assessment 363 of coronaviruses in CNS diseases with a focus on therapeutic targets

SARS-CoV-2 targets neurons of 3D human 366 brain organoids

Possible routes of SARS-CoV-2 invasion in brain: 368 In context of neurological symptoms in COVID-19 patients

The S1 protein of SARS-CoV-2 crosses the 371 blood-brain barrier in mice

Neuroinvasion of SARS-CoV-2 in human and mouse 373 brain

Neurologic manifestations of hospitalized patients with 375 coronavirus disease

Neuromechanisms of SARS-CoV-2: a review, 377 Front Neuroanat

Template-based protein structure prediction in CASP11 379 and retrospect of I-TASSER in the last decade

Control of cytolocalization and mechanism of cell 381 death by encapsulation of a photosensitizer

The effect of the D614G substitution on the 383 structure of the spike glycoprotein of SARS-CoV-2

Software update: the ORCA program system

AutoDock Vina: improving the speed and accuracy of docking with a 388 new scoring function, efficient optimization, and multithreading

Docking small peptides remains a great challenge: an assessment 391 using AutoDock Vina

Electrostatics of nanosystems: application to 393 microtubules and the ribosome

Interaction between albumin and zinc tetra-4-395

Investigation of interaction between 397 alkoxy substituted phthalocyanines with different lengths of alkyl residue and bovine serum 398 albumin

Spectral and hydrodynamic studies of complex 400 formation of tetraalkoxy substituted zinc(II)phthalocyanines with defatted and nondefatted 401 bovine serum albumin

The interaction of cationic and anionic 403 porphyrins with the bovine serum albumin in borate buffer

Features of interaction of tetraiodide 406 meso-tetra (N-methyl-3-pyridyl) porphyrin with bovine serum albumin

The interaction of 5

Composition and divergence of coronavirus spike proteins 412 and host ACE2 receptors predict potential intermediate hosts of SARS-CoV-2

SARS-CoV-2 and coronavirus disease 415 2019: what we know so far

TMPRSS2 and furin are both essential for 417 proteolytic activation of SARS-CoV-2 in human airway cells

Gold nanoparticle-adjuvanted S 420 protein induces a strong antigen-specific IgG response against severe acute respiratory 421 syndrome-related coronavirus infection, but fails to induce protective antibodies and limit 422 eosinophilic infiltration in lungs

Cell entry mechanisms of SARS-CoV-2

Receptor recognition by the novel coronavirus from 426 Wuhan: an analysis based on decade-long structural studies of SARS coronavirus

Mechanisms of host receptor adaptation by severe acute 429 respiratory syndrome coronavirus

Lifetime of oxygen (O2(1ΔG)) in liquid water as determined 431 by time-resolved infrared luminescence measurements

Spatially resolved cellular responses to singlet oxygen

Time-resolved singlet oxygen detection

Time-resolved singlet oxygen phosphorescence 438 measurements from photosensitized experiments in single cells: Effects of oxygen diffusion 439 and oxygen concentration

Architecture and self-assembly of the SARS-CoV-2 441 nucleocapsid protein

Biochemical characterization of SARS-CoV-2 nucleocapsid 443 protein

SARS-CoV-2 nucleocapsid protein 445 phase-separates with RNA and with human hnRNPs

Singlet oxygen-mediated damage to proteins and its consequences

The effects of photodynamic 450 treatment with new methylene blue N on the Candida albicans proteome

RNA 3'-end mismatch excision by the severe acute 453 respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease 454 complex

Structural basis and functional analysis of the SARS 456 coronavirus nsp14-nsp10 complex

The enzymatic activity of 458 the nsp14 exoribonuclease is critical for replication of MERS-CoV and SARS-CoV-2

A structural view of SARS-CoV-2 RNA 461 replication machinery: RNA synthesis, proofreading and final capping

Molecular docking established that hetaryl-substituted porphyrins form strong complexes with the S-, N-, and nsp13 proteins of SARS-CoV-2

Purposeful synthesis was carried out, and water-soluble unsymmetrical hetaryl-substituted porphyrins were obtained for the first time

The photochemical properties of unsymmetrically substituted hetarylporphyrins have been studied

Hetaryl-substituted porphyrins bind to the receptor-binding motif of the spike protein, which, along with a high quantum yield of single oxygen, has a virucidal action against SARS-CoV-2

The reported study was funded by RFBR, project number 20-04-60067. 360 361 J o u r n a l P r e -p r o o f

π-π-interaction between the porphyrin macroring and Tyr369 of the "A" chain (4 Å) H-bond between the N atom of the О-por hetaryl substituent and Trp415 of the "C" chain (3.6 Å) Spike protein -SPOR