Small molecules block SARS-CoV-2 spike-ACE2 attachment

The search for antiviral drugs and vaccines that can prevent cell invasion and infection caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues as the cold season in the northern hemisphere approaches, and further waves of COVID-19 Is threatening.

The search includes immunosuppressants, enzyme inhibitors, and soluble competitive inhibitors.Now, a new study published on the preprint server bioRxiv* In October 2020, provide preliminary evidence that small molecules can block viral invasion by blocking spike-ACE2 attachment.

Find a therapeutic target

For SARS-CoV-2 to invade host cells Spike protein A virus containing angiotensin converting enzyme (ACE2), which is a host cell receptor. This protein-protein interaction (PPI) is a potential target for antiviral drugs.

Antibodies are the first means of inhibiting PPIs, but a more direct approach is with smaller molecule inhibitors (SMIs), which are more robust to avoid and distort mutations. SMI is also susceptible to oral or inhalation administration, is less likely to provoke an immune response, and is easier to control.

Such attempts were made after the 2002-2004 SARS outbreak, and many researchers performed high-throughput screening (HTS) assays to identify agents that could block early viral invasion. doing. This included some SMI. But none of them do it for clinical development. This is primarily due to poor bioavailability, toxicity, and pharmacokinetics.

Researchers first screened small molecule libraries to identify new compounds that suppress spike-ACE2 interactions at low micromolar levels with compounds such as Congo Red and Evans Blue. Such dyes are excellent protein binders by default, demonstrating that their structure is designed as such, and providing a platform to help initiate such attempts.

The dye itself is usually not therapeutically useful, is a bright color, and in the case of azo dyes, tends to decompose rapidly. However, it can be chemically modified to overcome these shortcomings.

Inhibition of SARS-CoV-2 pseudovirus invasion

Based on this premise, researchers used this approach to find potential inhibitors of ACE2-S interactions between SMIs that could be developed therapeutically.Evidence that this is a valid approach comes from previous studies of other zoonotic diseases. Coronavirus..

The selected compound prevented pseudoviruses expressing the SARS-CoV-2-S protein from invading cells with ACE2 receptors at low micromolar levels at half the maximum inhibitory concentration (IC50). This may indicate that such organic dyes may provide a chemical scaffold for identifying novel PPI inhibitors.

The use of the protein thermal shift assay has shown that the identified SMI specifically binds to the SARS-CoV-2-S protein rather than the ACE2 molecule. Researchers also have in common in the presence of biphenyl linkers that have two aromatic groups at the ends (one naphthalene, the other aromatic naphthalene or phenyl) and have one or more polar substituents. I found a structural motif.

Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by the compounds of this study. Concentration-response curve test obtained for inhibition of PPI between SARS-CoV-2-RBD (His tagged, 0.5 μg / mL) and hACE2 (Fc binding, 1 μg / mL) in a cell-free ELISA type assay. Dye (A) and non-dye (B) compounds. The promiscuous PPI inhibitor erythrosine B (ErB) and the food coloring FD & C Yellow No. 6 (Sunset Yellow, SY) were included as positive and negative controls, respectively. The data are mean ± SD from two experiments and fit the standard sigmoid curve for IC50 measurements. Estimated IC50s are shown in the legend, where suramin and chloroquine were completely inactive (IC50> 500 μM), but in-house compounds containing organic dyes (CgRd, DV1, etc.) and proprietary DRI-C compounds (CgRd, DV1, etc.). Some of them, for example, DRI-C23041, DRI-CC91005) showed promising activity, even at submicromolar levels (IC50 <1 μM).

Importance of proper chemical space

The success of this experiment was due to the initiation in different chemical spaces, ensuring that the selected compounds were already effective at low micromolar concentrations. Unlike other recent studies that showed 25 possible hits of small molecules that disrupt ACE2-S binding, current experiments focus on molecules that are already known to have strong binding to proteins. I matched it.

Interestingly, none of the compounds identified as potential inhibitors in previous studies show the general chemical scaffolding described above, and it is known that a known drug or related compound is the SMI of PPI. It shows that it has a different structure than the one it has.

Benefits of SMI

An important advantage of SMI is their relative nonspecificity, which broadens the target of antiviral activity. In addition, they can even neutralize several types of viruses, regardless of antigen, through their unique mechanism of action. This is very important from the perspective of an economical therapeutic approach.

Again, the original purpose of the chemical structure of the developed drug was to regulate the CD40-CD40L co-stimulatory interaction with low micromolar efficacy. Some are selective, others are not so many. The discovery that they also inhibit ACE2-SPPI shows non-specific activity and may still be useful.

The SMI identified in the current study inhibited the formation of the ACE2-S complex and showed comparable efficacy in human and mouse tissues.

This may identify SMIs with a wide range of effects, including SARS-CoV, MERS-CoV, and other human coronaviruses, as the SMI targets in the current study were spike proteins of both SARS-CoVs. It shows that. SARS-CoV instead of -2 and host-specific ACE2. This suggests that it may block infection by both these viruses and some other coronaviruses.

Wide spectrum of antiviral activity

The SMI here is medium in size, but much smaller than what is usually identified as PPISMI. Increased size guarantees sufficient activity. Interestingly, they tend not to comply with the widely cited “five laws” criteria for effective medicines to ensure high bioavailability and good pharmacokinetics.

One of these is that the molecular weight (MW) must be less than 500. Many recently developed drugs likewise show a fundamental deviation from these criteria. The researcher said,Our results provide further evidence of the feasibility of SMI for CoV attachments and provide the first map of the chemical space needed to achieve this... “

Researchers have described their potential discoveries as follows:It is unique in pursuit of dual-function molecules with antiviral activity (eg SARS-CoV-2-S–ACE2) by inhibiting the interactions required for CoV attachment on the one hand and immunomodulatory activity on the other hand. Suppresses overt inflammation (inhibits CD40–CD40L) or releases the cytotoxicity of T cells to virus-infected cells (inhibits PD-1–PD-L1)The latter is especially important. Because T cell balance and recovery allows for proper viral clearance, prevents immune dysregulation, Cytokine storm It is often associated with the progressive disease of COVID-19.

Conclusion

Therefore, researchers have come up with several SMIs with promising inhibitory activity against S-ACE2PPI. This effect was tested against spike-expressing pseudovirions on cells expressing ACE2 and was found to be dose-dependent. Further development may result in alternative therapies for COVID-19 that can be used orally or by inhalation for treatment and prevention.

*Important Notices

bioRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.

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