Gold Metallodrugs to Target Coronavirus Proteins: Inhibitory Effects on the Spike‐ACE2 Interaction and on PLpro Protease Activity by Auranofin and Gold Organometallics**

Abstract Gold complexes have a long tradition in medicine and for many examples antirheumatic, anticancer or anti‐infective effects have been confirmed. Herein, we evaluated the lead compound Auranofin and five selected gold organometallics as inhibitors of two relevant drug targets of severe acute respiratory syndrome coronaviruses (SARS‐CoV). The gold metallodrugs were effective inhibitors of the interaction of the SARS‐CoV‐2 spike protein with the angiotensin converting enzyme 2 (ACE2) host receptor and might thus interfere with the viral entry process. The gold metallodrugs were also efficient inhibitors of the papain‐like protease (PLpro) of SARS‐CoV‐1 and SARS‐CoV‐2, which is a key enzyme in the viral replication. Regarding PLpro from SARS‐CoV‐2, the here reported inhibitors are among the very first experimentally confirmed examples with activity against this target enzyme. Importantly, the activity of the complexes against both PLpro enzymes correlated with the ability of the inhibitors to remove zinc ions from the labile zinc center of the enzyme. Taken together, the results of this pilot study suggest further evaluation of gold complexes as SARS‐CoV antiviral drugs.

The current pandemic outbreak of the severe acute respiratory syndromec oronavirus 2( SARS-CoV-2) has causeda nu nprecedented global health crisis with to date more than 29 million infected individuals. [1,2] While the world strugglesw ith the control of the fast outspreado ft his coronavirus and it's enormous impact on healthcare, economy and society,e fforts to develop vaccines and therapeutics have been undertaken worldwide at ar ate, which modernd rug discoveryh as not witnessede ver. The lack of an effective antiviral drug for the treatmento ft he Coronavirus disease-2019 (COVID-19) has triggered major drug repurposing efforts;h owever, to this date no approved therapeutic has proven to have sufficient efficacy in the many ongoing clinical trials. The urgentd evelopment of new innovative drug candidates against SARS-CoV-2 is the most important mission that medicinal chemists are currently facing.
Regarding drug activity evaluation, several molecular pathways have been in the focus of the search for ap ossible COVID-19 treatment based on strategies that had already been considered for the SARS-CoVand Middle East respiratory syn-dromeM ERS-CoV outbreaks. [3] Amongst others these include the entry of the coronavirus into the hostc ell (e.g. the interaction of TMPRSS2 [4] or ACE2w ith spike proteins of the coronavirus [5] ), the viral replication process in the host cell (e.g. the pro-teases3 CLpro [6] and PLpro [3,7,8] ), transcription,t he nucleocapsid protein, or exocytosiso ft he new virion. [3,7,9] Gold complexes have al ong lastingh istory in medicine and have been used as disease modifying antirheumatic drugs (DMARDs) for the treatment of rheumatoid arthritis. Intensive researcho no ther possible therapeutic applications of the lead compound Auranofin and other gold species has focused on anticancer and anti-infective agents. The applicationo fg old complexes as antiviral drugs has not been studied very intensively,a lthough some promising results suggest ap ossible futureu se as humani mmunodeficiency virus (HIV)t herapeutics. [10] Here we report the resultso fapilot study,i nw hichw ei nvestigated the effects of Auranofin and selected experimental gold metallodrugs( see Figure1)o nt wo relevant coronavirus targets (spikep rotein, papain like protease, PLpro). Whereas Au-1, [11] Au-3 [12,13] and Au-5 [14] were selected from our previous workso no rganometallic gold metallodrugs, Au-2 and Au-4 have not been reported before and their synthesis and characterization are described here. Complexes Au-1 to Au-5 are organometallics containinge ither a N-heterocyclicc arbene (NHC) or an alkynyll igand.C omplexes of these types have demonstrated promising activities in af ast increasing number of recentr eports. [15] The entry of SARS-CoV-2 into target cellsi sf acilitatedb yt he spike (S) protein of coronaviruses and mediated by the angiotensin-converting enzyme 2( ACE2)a st he entry receptor. [1,4] The S1 subunit of the SARS-CoV-2 spike protein contains the receptor binding domain (RBD). Binding of the RBD to the human ACE2 receptor can be measured by ELISA allowing to evaluatei nhibitors of the Sp rotein ACE2 interaction. In this assay,t he gold complexes Au-1 to Au-5 and Auranofin displayed good IC 50 values in the range of 16-25 mm and were thus slightly more active than the reference drug Chloroquine (IC 50 value:31.9 mm).
An essential step in the replication of coronaviruses is the processing of the replicase polyprotein by proteases, such as the papain-like protease (PLpro), resulting in an umber of nonstructural proteins (nsps) that are involved in downstream binding and replicatione vents. [3,7,8] SARS-CoV-1P Lpro shares 83 %s equence identity with PLpro from SARS-CoV-2, structural components of the active sites of the enzymes do not substantially differ.A sac ysteine protease PLpro is al ikely target for gold-based drugs, which generally are known to interact with sulfur-containing molecular targets.
The inhibitory activity of the gold compounds towards PLpro from SARS-CoV-1 and SARS-CoV-2 was determined by an enzymatic FRET assay.A gainstP Lpro from SARS-CoV-1, Au-1, Au-2 and Au-5 exhibited IC 50 valuesi nt he range of 5-7 mm matching the potency of the reference inhibitor Disulfiram. Complexes Au-3 and Au-4 were less active with IC 50 valueso f 14 mm.A uranofin remained the lowest active gold compound with an IC 50 value of 25.5 mm (Table 1).
The missing activity of Au-3 and Au-4 against SARS-CoV-2 PLpro andt heir lower activity against SARS-CoV-1 PLpro com-pared to the other gold compounds indicates that the absence of the more easily exchangeable chlorido, and phosphane ligands prevents as tronger interaction of the gold center with the enzyme. It should also be noted that complexes Au-3 and Au-4 with their moderate activity against SARS-CoV-1 PLpro followed the opposite trend than the other compounds, which were more active against SARS-CoV-2 PLpro than against the enzymef rom SARS-CoV-1.
Cysteine residues in both types of studied SARS-CoV PLpro are the likely binding sites for gold metallodrugsa nd this interaction will be facilitated by ligand replacement reactions at the gold center. Importantly,apreprint report confirms that the catalytic cysteine 111 in the active site of SARS-CoV-2 PLpro can engage in Michael addition reactions with the b-carbono f vinyl groups of inhibitors. [16] Coordinationo ft he gold center to Table 1. Inhibition of the spike-ACE2 interaction and PLpro activity (mean valuesand standard deviations, n = 3-4);n.d. not determined.Benzimidazole was used as anegative reference in both assays.  Besides the catalytic cysteine PLpro hosts several cysteine residues in ap utative labile Zn-binding domain, whichi sr esponsible for correctf olding of the protein and stabilizationo f the local geometry.E jection of Zn 2 + from this site represents another likelym echanism for inhibition of PLpro. [17] Of note, the replacement of zinc from zinc-finger motifs and formation of so called gold-fingersh as been well documented. [18] Interestingly, av ery recent paper reportso nt he dual activity of thiol-reacting inhibitor Disulfiram as zinc removing agent as well as modifier of the catalytic cysteine of SARS-CoV-2 PLpro. [19] As Disulfiram displayed similar activity with the Au-1, Au-2 and Au-5 against both types of PLpro, it could be speculated that these compounds share such dual activity against the enzyme. Thus we evaluated the ability of the inhibitors to replacez inc ions from PLpro by measuringt he released zinc with azinc selective fluorescent dye.
In the experiments with SARS-CoV-1 PLpro, the most efficient inhibitors Disulfiram, Au-1, Au-2 and Au-5 were effective zinc ejectors,w hile Auranofin as the lowesta ctive enzymei nhibitor was not an efficient zinc ejector.T he zinc removing activity of the moderate SARS-CoV-1 PLpro inhibitors Au-3 and Au-4 was strongly time-dependent.
In the studies with SARS-CoV-2 PLpro all compounds except Au-3 and Au-4 were efficient zinc ejectors (Figure 2). These re-sults are in excellent agreement with the inactivity of Au-3 and Au-4 against SARS-CoV-2 PLpro as well as the high activity of Auranofin against this enzyme. Ta ken together, the results of the zinc ejection experiments correlate very well with the activity profile of the gold complexes in the enzymatic FRET assays and explain the differing activitieso ft he complexes against the two types of PLpro.
In conclusion, we have demonstrated that gold complexes can target two relevant pathways in the life cycle of coronav iruses. The strongesta ctivity was noted against SARS-CoV-2 PLpro with Auranofin and the organometallic gold complexes Au-1, Au-2 and Au-5.T he compounds belongt hus to the very first potent inhibitorso ft he target enzyme. Theira ctivity against the enzymec orrelates very well with their zincejectinge fficacy.
Notably,t he inhibition of the replication of SARS-CoV-2 by Auranofin in human cells at low micro molarc oncentration (below the IC 50 value for cytotoxicity)w as reportedv ery recently. [20] For Au-1, Au-3 and Au-5 strongc ytotoxic activity and effectso nt he cellular signaling have been reported previously. [11,12,14] Such strong effectso nh ost cells at this stage would hamper accurate characterization of possible antiviral effects in cell based models. Hence, ad esirable reduction of cytotoxicity againsth ost cellss hould accompanyt he ongoing target identification and structure optimization efforts.
The screening of gold and other metal-based drugs towards relevant SARS-CoV-2 molecular targets in combination with a toxicitye valuationi sd efinitely warranted and is ongoing in our laboratories.

Spike/ACE2 interaction assay
The inhibition of the spike-ACE2 interaction was measured using the SARS-Cov2 Inhibitor Screening Assay kit (Adipogen, Cat. N8 AG-44B-0007-KI01). All reagents were used from the same kit during individual experiments and the experiment was performed using the manufacturer's protocol. Briefly,t he SARS-CoV-2 Spike S receptor binding domain (RBD):Fc (human) (rec.) (SPIKE) was reconstituted to 0.1 mg/mL with deionized water.T his was further diluted to aw orking concentration of 1 mg/mL in phosphate buffered saline (PBS) and used freshly.T he assay plate was coated with 100 mL/well of SPIKE, covered with ap lastic film and kept at 4 8C overnight. The liquid was aspirated and any remaining liquid was removed by blotting against clean absorbent papers. The plate was blocked using 200 mLo fB locking Buffer per well for 2h at room temperature. The liquid was aspirated and the wells were washed with 1X Washing Buffer (300 mL x 3t imes). All liquid was aspirated and excess liquid was removed by blotting against clean absorbent papers. The inhibitors (gold complexes, controls, reference) were diluted in Inhibitor Mix Solution (IMS), which was prepared using ACE2 (human) (rec.) (Biotin) (ACE2) (0.1 mg/mL) to the working concentration of 0.5 mg/mL in 1X ELISA Buffer.T he stock solution of the inhibitors was made in DMSO and the final DMSO concentration in the wells was 0.5 %. The IMS-diluted inhibitors were added to the wells (100 mL/well). The final concentrations of the inhibitors were in the range of 1t o1 00 mm.T he negative control wells were also treated with 0.5 %D MSO in IMS. The plate was covered with ap lastic film and incubated at 37 8Cf or 1h after which the aspiration/ wash step was repeated. Next, horseradish peroxidase-labeled streptavidin (HRP) was reconstituted with 100 mLo f1 XE LISA Buffer and further diluted to aw orking concentration by adding 50 mLi n1 0mLo f1 XE LISA Buffer (1:200 dilution). It was covered with ap lastic film and incubated at RT for 1h.
Following this, the aspiration/ wash step as described earlier was repeated. Substrate development was conducted by the addition of 100 mLo fr eady-to-use 3,3',5,5'-tetramethylbenzidine (TMB) to each well for 5m inutes at RT.T he reaction was stopped by adding 50 mLo fastop solution. The OD was measured at 450 nm using a PerkinElmer Victor X4 microplate reader.T he individual absorbance value of the blank well was subtracted from the other absorbance values and the percentage of the remaining activity was calculated with respect to the untreated control values. Data fitting was done using Origin 2018 using sigmoidal fitting with Hill1 fitting curve. All treatments were done in duplicates and two independent experiments were performed.

SARS-CoV-1 and SARS-CoV-2 PLpro inhibition
The inhibition of PLpro was determined according to reported protocols with minor modifications. [23] The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mm HEPES, pH 7.5, 0.1 mg mL À1 bovine serum albumin, 0.1 %T riton-X100) to micromolar concentrations. Volumes of 50 mLo f3 50 nm His 6 -SARS-CoV-1 PLpro (SouthBayBio) or of 200 nm SARS-CoV-2 PLpro (Elabscience) in HEPES buffer or blank HEPES buffer (negative control) were added to the wells of ab lack 96-well microtiter plate (Nunclon, Nunc). Volumes of 50 mLo ft he inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (175 nm SARS-CoV-1 PLpro or 100 nm SARS-CoV-2 PLpro 0.5 %D MSO, 0.1-100 mm test compound or blank HEPES buffer) were mixed and incubated at 37 8Cf or one hour.Av olume of 100 mLo f1 00 mm Z-Arg-Leu-Arg-Gly-Gly-AMC (Bachem Bioscience) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every 30 sf or 10 min (l exc = 355 nm; l em = 460 nm) at 37 8C using aV ictor TM X4 Perkin Elmer 2030 multilabel reader.T he increase of emission over time followed al inear trend (r 2 > 0.97) and the enzymatic activities were calculated as the slope thereof. The IC 50 values were calculated as the concentration of the inhibitor that was required to decrease the enzymatic activity to 50 %o ft he positive control. The wells containing the negative control were used to confirm the absence of false positive results by reaction of the inhibitor compound with the fluorogenic substrate.

Zn-ejection assays with SARS-CoV-1a nd SARS-CoV-2 PLpro
To determine if the inhibitors are Zn-ejecting agents the presence of the Zn 2 + cation in solution was measured according to arecently published preprint. [19] The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mm HEPES, pH 7.5) to 100 mm concentrations. Volumes of 50 mLo f1mm His 6 -SARS-CoV-1 PLpro (SouthBayBio) or SARS-CoV-2 PLpro (Elabscience) in HEPES buffer or blank HEPES buffer (control for false positive results) were added to the wells of a black 96-well microtiter plate (Nunclon, Nunc). Volumes of 50 mLo f the inhibitor solutions or 1% DMSO in HEPES buffer (control) were added. The resulting solutions (500 nm PLpro SARS-CoV-1 or PLpro SARS-CoV-2, 0.5 %D MSO, 50 mm test compound or blank HEPES buffer) were mixed. Av olume of 100 mLo f2 .0 mm of zinc-specific fluorophore FluoZin TM -3 (Invitrogen/LifeTechnologies) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured after 10 min every 10 min for 90 min (l exc = 485 nm; l em = 535 nm) at 37 8Cu sing aV ictor TM X4 Perkin Elmer 2030 multilabel reader.T he relative fluorescence was calculated by dividing the absolute fluorescence emission of the well containing the inhibitor by the absolute fluorescence of the respective well containing the enzyme but no inhibitor (control).