International teams join forces to accelerate drug design based on the structure of COVID-19!

SARS-CoV-2 is an enveloped single-stranded RNA virus. The viral RNA produces two polyproteins: pp1a and pp1ab through ribosome frameshift. The polyprotein is processed by the papain-like protease (PL pro), which cuts 3 sites to release the non-structural protein nsp1-3 and a 3C-like protease, also known as the main protease (M pro). Cut at 11 sites to release non-structural proteins. These non-structural proteins form a replicase complex and are responsible for the replication and transcription of the viral genome. Therefore, M pro and PL Pro have become the main targets of antiviral drug development.

Structural biology can play a key role in drug development, but drugs that directly target SARS-CoV-2 are still elusive. Recently, a new paper in “Nature Communications” outlines how an international research team has identified potential methods to quickly design improved and more powerful compounds against COVID-19. The researchers performed a large-scale electrophile and non-covalent fragment screening virus replication on the SARS-CoV-2 main protease by combining mass spectrometry and X-ray methods. The crystallography screen identified 71 matches and two 3 matches at the polymer interface. These combinations provide unprecedented structure and reactivity information, which can be used for ongoing drug design targeting the structure of the SARS-CoV-2 main protease.

Part 1
M pro crystallizes in a ligand-free form and diffracts to near atomic resolution

The crystal structure of M pro without ligand is suitable for X-ray fragment screening (Source: Nature Communications)
The crystal form is very suitable for the screening of crystal fragments. Although the solvent percentage of protein crystals is very low, there are still clear channels through which diffusion can enter the active site. In addition, when soluble fragments are added to the crystal droplets, the tightly packed and firm innate diffraction average crystals can resist the destruction of the crystal lattice and the degradation of diffraction by the DMSO solvent.

Part 2
Combining MS and crystallographic fragment screening reveals a new binder for M pro

In order to determine the covalent starting point, the researchers used the complete protein profiling method to screen the previously described library of approximately 1,000 mildly electrophilic fragments against Mpro. Containing the compound N-(chloroacetyl)aniline motif is a frequent hitter because such compounds are highly reactive. Therefore, the researchers selected series members with relatively low reactivity for subsequent crystallization attempts. For another series of hit compounds containing the N-chloroacetylpiperidinyl-4-carboxamide motif, they showed low reactivity and Strikes did not occur frequently in previous screenings.
Although mild electrophilic fragments are very suitable for probing the binding properties around the active site cysteine, their small size prevents extensive exploration of substrate binding. The researchers conducted another crystallographic fragment screening to explore the active sites of Mpro in detail and look for opportunities for fragments to merge or grow. 68 electrophilic fragment hits and a total of 1176 unique fragments from 7 libraries were added to the crystal, non-covalent fragments were soaked, and electrophilic fragments were both soaked and co-crystallized to ensure structural observation As many mass spectra as possible. A total of 1742 immersion and 1139 co-crystallization experiments produced 1877 mosaic crystals. Although some fragments destroyed the crystals or diffraction, 1638 data sets with a resolution higher than 2.8Å were still collected.

Part 3
Non-covalent fragment hits reveal multiple targetable subsites in the active site

The researchers identified eight fragments that bind to the S1 subsite, and interact with the side chains of key residues through the pyridine ring or similar nitrogen-containing heterocycles, and interact with Glu166 through the carbonyl group in the amide or urea moiety. effect. Compared with other subsites, subsite S2 has previously shown greater flexibility, adapting to smaller substituents in peptidyl inhibitors, but preferentially choosing leucine or other hydrophobic residues. Many fragments bind at this position. Researchers call it the “aromatic wheel” because the consistent motif of the aromatic ring forms a hydrophobic interaction with Met49, or forms a π-π stacking with His41, and the groups are in 4 axial directions. place.

In exploring the four fragments of subsite S3, three of them have an aromatic ring of a sulfonamide group. The sulfonamide group forms a hydrogen bond with Gln189 and points to the active site toward the solvent interface. These hits are suitable Use the same expansion vector of His164 / Met165 / Asp187 pocket as mentioned above.
It has been determined that the biological unit of similar viral proteases such as SARS-CoV-1 protease is a dimer, and the protease activity can be destroyed at the mutant dimer interface, so compounds that interfere with dimerization may serve as a standard for protease activity. Allosteric inhibitor. In this study, the three compounds are bound at accessible locations on the dimer interface, and it is conceivable to use them to design compounds to destroy the M pro dimer.

Fragment Z1849009686 (×1086) is bound in a hydrophobic pocket formed by the side chains of Met6, Phe8, Arg298 and Val303. It also mediates two hydrogen bonds with the side chains of Gln127 and the backbone of Met6. Z264347221 (×1187) is similarly bound in a hydrophobic pocket made of Met6, Phe8 and Arg298, extending across the dimer interface to interact with Ser123, Tyr118 and Leu141 of the second primary protein, including hydrogen Key to the side chain and backbone of Ser123. Finally, POB0073 (x0887), which only binds to Gly2 4Å at the dimer interface, is wrapped between Lys137 and Val171 of one precursor, and the other is wrapped between Gly2, Arg4, Phe3, Lys5 and Leu282, including Two hydrogen bonds connected to the Phe3 skeleton.

Part 4
Covalent fragment hits reveal several manageable series

In all structures with electrophilic binding, the N-chloroacetylcarbonyl oxygen atom forms two or three hydrogen bonds with the backbone amide hydrogen of Gly143, Ser144 or Cys145, including N-chloroacetylpiperidinyl-4- All three compounds of the carboxamide motif use similar binding methods that point to the S2 pocket.

A series of compounds containing the N-chloroacetylpiperidinyl-4-carboxamide motif showed promising binding modes. In order to track these compounds, the researchers conducted rapid synthesis of second-generation compounds. By reacting N-chloroacetylpiperidine-4-carbonyl chloride with various internal amines (preferably with chromophores to simplify purification), this chemical type of derivative can be obtained in milligrams. These new compounds are tested by intact protein mass spectrometry to evaluate protein markers. Amides derived from non-polar amines mostly outperform their polar counterparts, which suggests a lipophilic subregion that can be targeted in this direction. The two highest labeled amides of PG-COV-35 and PG-COV-34 highlight the potential for further synthetic derivatization by amide N-alkylation or cross-coupling, respectively.
The highlighted structure-activity relationship is important for further optimization. Bromoacetylene has inherent thiol reactivity, which is lower than the inherent thiol reactivity of established acrylamide-based covalent inhibitors. The geometry of alkynes and their combination also indicate that they can be replaced by reversible covalent groups (such as nitrile), which can be guided by the same non-covalent interactions. Two covalent hits (2-cyanopyrimidine and 2-cyanoimidazole) come from a small heterocyclic electrophile library. They are essentially composed of five-membered and six-membered nitrogen and contain heterocyclic rings with electron-withdrawing properties. Can activate small electrophilic substituents (halogen, ethyl, vinyl and nitrile).

Part 5
Results and discussion

The data provided in this study provides many clear ways to develop effective M pro inhibitors from SARS-CoV-2. The bound fragments comprehensively sample all sub-sites of the active site, reveal different amplification vectors, and electrophiles provide systematic and accidental extensive data for the design of covalent compounds. It is generally believed that new small molecule drugs cannot be developed fast enough to help fight COVID-19. However, since the threat of a pandemic will still be a long-term problem, and candidate vaccines cannot guarantee comprehensive and lasting protection, antiviral molecules will remain an important line of defense. Such compounds will also need to fight future epidemics. The data from this study will accelerate the progress of such work.
In general, covalent hits provide a reasonable approach to low-reactivity and highly selective inhibitors. Reasonably designed covalent drugs gain traction. Many of the drugs approved by the FDA recently are designed based on very effective non-covalent binding, which allows precise positioning of low reactivity and electrophilicity, so the formation of covalent bonds is the binding site Point-specific dependence. Fragmentation method has become a main method of modern drug discovery, using small collections (100s or 1000s) of a small number of compounds (<300Da), these compounds are disorderly combined, so the chemical space obtained by HTS is much larger. The challenge is that the combination of fragment hits is very weak, and highly sensitive biophysical detection, careful confirmation of the combination and professional chemical knowledge are needed to hit and develop these into fully effective drug candidates. But the real hope is that they can be quickly and effectively converted into effective drug candidates, and they can be converted into clinical drugs in a simpler way.
The world’s focus has always been on the reuse of vaccines and existing drugs, but this research is one of the few projects to try new small molecule therapies. The research team took a very unusual approach, which is to publish all experimental data immediately after it is generated, so the results will be available to any drug manufacturer in the world. The international community’s response to the real-time release of this research data has been even better, mobilizing a large amount of professional knowledge, technology and charity, and has evolved into a unique and rigorous drug discovery work aimed at rapid development of good safety and clinical The original oral antiviral drugs. Medicilon, as a new drug research and development CRO, will continue to pay attention to this research progress.

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