key: cord-336605-d4loia11
authors: Zhang, Xue Wu; Yap, Yee Leng
title: Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs
date: 2004-05-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2004.03.035
sha: 
doc_id: 336605
cord_uid: d4loia11

The SARS-associated coronavirus (SARS-CoV) main proteinase is a key enzyme in viral polyprotein processing. To allow structure-based design of drugs directed at SARS-CoV main proteinase, we predicted its binding pockets and affinities with existing HIV, psychotic and parasite drugs (lopinavir, ritonavir, niclosamide and promazine), which show signs of inhibiting the replication of SARS-CoV. Our results suggest that these drugs and another two HIV inhibitors (PNU and UC2) could be used as templates for designing SARS-CoV proteinase inhibitors.

Reemergence of severe acute respiratory syndrome (SARS) is a distinct possibility. Currently neither antiviral therapy nor vaccine is available. Viral replicase and protease are preferred targets for the screening and design of antiviral compounds and have been successfully targeted in several viral diseases. The SARS-associated coronavirus (SARS-CoV) main proteinase (Mpro or 3CL pro) plays a key role in proteolytic processing of the replicase polyproteins 1a and 1ab, which makes it an attractive target for developing drugs against this new disease. Recent report indicated that the proteinase inhibitor kaletra, a mixture of protease inhibitors-lopinavir and ritonavir, approved for treating HIV in 2000, shows signs of effectiveness against the SARS virus. 1 In particular, researchers in Taiwan discovered that two existing medicines, which have significant effect in inhibiting the replication of SARS-CoV (http://www. etaiwannews.com/Taiwan/2003/10/31/1067562739.htm). One is an anti-parasite drug niclosamide, and another is anti-psychotic drug promazine. The purpose of this study is to analyze whether the SARS-CoV main proteinase could be the target of these existing drugs. We performed in silico binding studies of the drugs using the recently identified crystal structure of Mpro, 2;3 to provide information for anti-SARS inhibitor design.

The atomic coordinates of SARS-CoV main proteinase were downloaded from Protein Data Bank (PDB ID 1Q2W). Another crystal structure of SARS-CoV main proteinase is also available (PDB ID 1UJ1), the superposition of 1Q2W A chain and 1UJ1 A chain is shown in Figure 1 , they overlap very well (rmsd ¼ 0.64), here we chose 1Q2W as docking studies, which was released early. The overall structure of a monomer of SARS-CoV main proteinase is composed of three domains: domain I (residues 1-101), domain II (residues 102-200) and III (residues 201-303), represented by green, pink and white trace in Figure 1 . The cleft between domains I and II is its substrate-binding site.

Except four drugs (lopinavir, ritonavir, niclosamide and promazine), we also conducted the docking studies of two other molecules, PNU and UC2, for their molecular formulas are close to those of niclosamide and promazine, respectively (Fig. 2) , and they both are the inhibitors of HIV-1 reverse transcriptase. 4;5 The program Hex was employed to conduct the docking of the ligands to the SARS-CoV main proteinase, its basic approach to parametric functions, which encode both surface shape, electrostatic charge and potential distributions. The surface shape representation uses a novel 3D surface skin model of protein topology, and a novel soft molecular mechanics energy minimization procedure is used to refine the candidate docking solutions. Unlike conventional 3D fast Fourier transform (FFT) docking approaches, Hex uses spherical polar Fourier correlations to accelerate the docking between 10 and 100 times faster than FFT docking algorithm. 6 Here we used the following parameter set: correlation type ¼ shape + three probes, post-processing ¼ MM minimization, steric scan ¼ 20 (maximum), final search ¼ 32 (maximum), the others are default set. The structural comparison was performed by LGA. 7 The visualization of 3D structure was generated by PROTEINEXPLORER (http://www.proteinexplorer.org). Figure 3 displays the overall structures of docking for four drugs (lopinavir, ritonavir, niclosamide and promazine) and two inhibitors (PNU and UC2) to SARS-CoV main proteinase. The binding pockets of these compounds in SARS-CoV main protease are shown in Table 1 , which is defined by those residues that have at least one heavy atom (other than hydrogen) with a distance less than 5

A from a heavy atom of inhibitors, as described by Chou et al. 8 The results show that the binding pockets of six compounds can be divided into three classes: (1) residues 40-86 and 181-192 for four drugs/inhibitors (lopinavir, niclosamide, promazine and PNU); (2) residues 41-51 and 164-194 for UC2 inhibitor; (3) residues 19-57 and 117-193 for drug ritonavir. All these pockets locate in domain I (residues 8-101), domain II (residues 102-184) and a long loop region (residues 185-200) connecting domains I and II in SARS-CoV main proteinase. Thus, the four drugs and two inhibitors studied here can basically bind to the active site of SARS-CoV main proteinase, a cleft between domains I and II.

To estimate the binding affinities of each compound, the inhibitory constant (K i , mole) was calculated from the equation:

where DG is the free energy of binding (kJ/mol) (here refers to the final docked energy), R is the gas constant 8.31 J/K/mol and T is the absolute temperature (at 300 K), as did in Jenwitheesuk and Samudrala. 9 The results indicate that the inhibitory constants of six compounds are: 8.7 · 10 À20 (lopinavir), 5.6 · 10 À25 (ritonavir), 4.2 · 10 À22 (niclosamide), 6.2 · 10 À21 (promazine), 3.5 · 10 À23 (PNU), 2.1 · 10 À19 (UC2). It is noted that these values are too low, for example, the inhibitory constant of lopinavir was determined as $10 À7 by Jenwitheesuk and Samudrala. 10 The reason for this difference is that the docked energy value from Hex program is a pseudo-energy, which is designed to give reasonably consistent units with conventional energy calculations, not based on experimentally derived parameters, and as a theoretical reference value only when performing the docking algorithm. Thus we do not expect these values are the genuine representations of inhibitory constants and we use them primarily for qualitative comparison among the drugs/inhibitors studied here. Because the lower the K i is, the greater the binding affinity is, hence HIV drug ritonavir is the compound that bind to the substrate binding site of SARS-CoV proteinase with the highest binding affinity, followed by HIV inhibitor PNU and anti-parasite drug niclosamide, and UC2 is the compound with the lowest binding affinity. Moreover, the inhibitory constants of ritonavir, PNU, niclosamide, promazine and UC2 are about 10 À5 , 10 À3 , 10 À2 , 10 À1 and 10-fold inhibitory constant of lopinavir, respectively, if we assume that a value of 10 À7 mol for lopinavir's inhibitory constant is correct, the inhibitory constants of ritonavir, PNU, niclosamide, promazine and UC2 could be estimated as 10 À12 , 10 À10 , 10 À9 , 10 À8 and 10 À6 mol, respectively.

The close views of the interactions between SARS-CoV main proteinase and these drugs/inhibitors are exhibited in Figure 4 . The results show that half of lopinavir is left outside the catalytic site (Fig. 4a) , for ritonavir, the thiazole group (P1) and a benzene group (P2) are inserted into S1 and S2 specificity pockets, respectively, while another benzene side chain (P3) might be too long to fit the substrate binding pocket perfectly (Fig. 4b) , there is similar situation in the inhibitor AG7088, 11 which has been experimentally shown to not bind with high affinity to the SARS-CoV proteinase (http://www.nature.com/ nsu/030512/030512-11.html). Thus the efficacy of lopinavir/ritonavir could be poor. Indeed, consistent with our predictions, experimental observation data indicated that both lopinavir and ritonavir individually have only a weak in vitro activity against SARS-CoV. However, the addition of lopinavir/ritonavir to ribavirin and corticosteroid treatment regimens appears to reduce incubation and mortality rates, especially when administered early. 12 Similarly, the half of niclosamide or promazine is left outside the active site ( Fig. 4c and d) , obviously the propane side chain in promazine is too long. For PNU inhibitor, seems it can basically fit into the active cleft, except the dihydrofuran side chain is a little bit long (Fig.  4e) . Finally, the inhibitor UC2 binds to a position that is slightly away from the active centre (Fig. 4f) , its neopentane or methylfuran side chain is a little long and makes it unable to insert into the active pocket properly. Indeed UC2 is the compound with lowest binding affinity as mentioned above. Taken together, our study illustrates that existing drugs/inhibitors may be used as starting points for the discovery of rationally designed anti-SARS proteinase drugs. 

Old drugs for a new bug

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

Crystal structures of HIV-1 reverse transcriptase in complex with carboxanilide derivatives

Evaluation of protein docking predictions using Hex 3.1 in CAPRI rounds 1 and 2

LGA: a method for finding 3D similarities in protein structures

Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS

Improved prediction of HIV-1 protease-inhibitor binding energies by molecular dynamics simulations

Identifying inhibitors of the SARS coronavirus proteinase

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

Treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study