We have completed an initial screen of our in-house developed secondary metabolite library of over 3.5 lakh molecules for novel antivirals against a set of 6 viral targets. Further in-depth screens are underway.
This is an in-house proprietary project.
THE 6 TARGETS :
Let us take a detailed look into the 6 selected viral targets we are currently screening for
(note: all sequences based on Wuhan strain NC_045512.2)
There are 4 subunits of the Spike (S) protein. The Spike protein S1 subunit attaches the virion to the cell membrane by interacting with host receptor human ACE2 and CLEC4M/DC-SIGNR, initiating the infection. Spike protein S2 subunit mediates the fusion of the virion and cellular membranes by acting as a class I viral fusion protein. Spike protein S2′ subunit acts as a viral fusion peptide which is unmasked following S2 cleavage occurring upon viral endocytosis. Finally, the Spike protein subunit S3 houses the RBD (receptor binding domain). We have targeted this subunit S3 in our screening studies.
It is a viral envelope component and plays a central role in virus morphogenesis and assembly via its interactions with other viral proteins. A homo pentamer, it Interacts with membrane protein M in the budding compartment of the host cell, which is located between endoplasmic reticulum and the Golgi complex. It also interacts with human MPP5 thus inhibiting the interaction between human MPP5 and human CRB3, further causing delayed tight junction formation and defective cell polarity.
Active transport of proteins from the cytoplasm to the nucleus is mediated by a family of nuclear transport receptors known as importins (or karyopherins) of which there are 7 isoforms. They act together with a number of ancillary proteins including nucleoporins and Ran via the classical nuclear import pathway. After initiation by Importin α, The cargo-IMPα complex gets transported through the nuclear pore complexes (NPCs) by building heterotrimer complex with importin β, necessitating interactions with FG repeat regions on nucleoporin proteins.
The viral 3-chymotrypsin-like cysteine protease (3CLpro) enzyme controls coronavirus replication and is essential for its life cycle. 3CLpro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERSCoV).
Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV. 3CLPro interacts with the C-terminus of the Replicase polyprotein.
Responsible for the cleavages located at the Nterminus of the Replicase polyprotein, PL-PRO also possesses a deubiquitinating activity and
further participates in the assembly of virally induced cytoplasmic double-membrane vesicles necessary for viral replication. It can antagonise the induction of type I interferon by blocking IRF3 and NFκB signalling. A comparison of the unbound and inhibitor-bound structures of PLPRO reveal 2 significant conformational differences. In the unbound structure, a highly mobile loop exists while binding to an inhibitor induces closure of this loop causing the side chains of Y269 and Q270 to become well-defined and reorient to close over the inhibitor. We have targeted Y269 and Q270 residues.
RNA-dependent RNA polymerase (RdRp, also known as nsp12), catalyses the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of COVID-19 virus, possibly with the assistance of nsp7 and nsp8 as co-factors. nsp12 is therefore considered a primary target for nucleotide analog antiviral inhibitors such as Remdesivir, which shows potential for the treatment of COVID-19 viral infections. The active site of the COVID-19 virus RdRp domain is formed by the conserved polymerase motifs A-G.We have targeted the motif C focused on catalytic SDD residues.
Human coronavirus 2019-nCoV (also known as SARS-CoV-2) is causing a pandemic with significant morbidity and mortality making it imperative for effective interventions with increasing urgency. With time being short and no effective novel drugs targeting SARS-CoV-2 available as yet, drug repurposing of existing drugs is emerging as the most efficient discovery based intervention strategy. The strategy can be beneficial because of its shortened discovery time, reduced cost and already clinically proven safety for human use. With these advantages in mind, we have performed an in silico drug repurposing study implementing successful concepts of computer aided drug design (CADD) technology against the first 2 SARS-COV-2 target proteins – the Spike (S) protein and the Envelope (E) protein.
We continue to work on identifying the suitability of these drugs against the remaining 4 viral target proteins described above.
We follow the same approach for our in-house plant based small molecule screening studies for novel antivirals. We have created a small molecule library of over 450,000 plant secondary metabolites. This library has now been screened for molecules with anti-SARS-CoV-2 activity.
First, We performed sequence analysis of the complete genome of SARS_nCoV-2 and identified 6 viral protein targets. These have been described in detail above.
We then performed homology modelling of the viral target protein structures to prepare them for docking studies with our in-house developed phyto-molecule library. Then, molecular docking was performed using Glide application in two stages with three levels of precision to generate highly accurate in silico HITS/LEADS. Our library of close to 5 lakh molecules has been whittled down based on their chemical nature and predicted safety in humans, to generate a list of molecules that we now intend to investigate further in in vitro and in vivo validation studies against the SARS-CoV-2 virus to confirm the safety and therapeutic efficacy of the molecules.
Encouragingly, since the molecules are present in plants, which have been safely used regularly in Ayurveda, the anti-viral activity of the plants can be rapidly established. Once safety is established, we can also formulate and commercialise the oral antiviral quickly.
The entry of SARS-CoV-2 into host cells is mediated by homotrimer, protruding and transmembrane SARS-CoV-2 S glycoprotein localised on viral surface. The total SARS-CoV-2 S is comprised of two functional subunits individually responsible for interaction with host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit).
Corona virus (CoVs), SARS-CoV-2 S is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the perfusion conformation. The receptor-binding domain (RBD) site on distal S1 subunit contributes to the stabilisation of the profusion state of the membrane-anchored S2 subunit that contains the fusion machinery.
For all CoVs, spike glycoprotein is further cleaved by host proteases at the so-called S20 site located immediately upstream of the fusion peptide.
This cleavage has been proposed to activate the protein for membrane fusion via extensive irreversible conformational changes. As a result, coronavirus entry into susceptible cells is a complex process that requires the concerted action of receptor binding and proteolytic processing of the SARS-CoV-2 S protein to promote the fusion of SARS-CoV-2 cells.