Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening.

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months1,2or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase.

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development.

Zika virus (ZIKV) infections cause microcephaly in new-borns and Guillain-Barre syndrome in adults raising a significant global public health concern, yet no vaccines or antiviral drugs have been developed to prevent or treat ZIKV infections. The viral protease NS3 and its co-factor NS2B are essential for the cleavage of the Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 48 binders with diverse chemical scaffolds were identified in the active site of the protease, with another 6 fragment hits observed in a potential allosteric binding site. Our work provides potential starting points for the development of potent NS2B-NS3 protease inhibitors. Furthermore, we have structurally characterized a potential allosteric binding pocket, identifying opportunities for allosteric inhibitor development.

Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals.

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16 . Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

Multiscale simulations reveal the role of PcrA helicase in protecting against spontaneous point mutations in DNA.

Proton transfer across hydrogen bonds in DNA can produce non-canonical nucleobase dimers and is a possible source of single-point mutations when these forms mismatch under replication. Previous computational studies have revealed this process to be energetically feasible for the guanine-cytosine (GC) base pair, but the tautomeric product (G[Formula: see text]C[Formula: see text]) is short-lived. In this work we reveal, for the first time, the direct effect of the replisome enzymes on proton transfer, rectifying the shortcomings of existing models. Multi-scale quantum mechanical/molecular dynamics (QM/MM) simulations reveal the effect of the bacterial PcrA Helicase on the double proton transfer in the GC base pair. It is shown that the local protein environment drastically increases the activation and reaction energies for the double proton transfer, modifying the tautomeric equilibrium. We propose a regime in which the proton transfer is dominated by tunnelling, taking place instantaneously and without atomic rearrangement of the local environment. In this paradigm, we can reconcile the metastable nature of the tautomer and show that ensemble averaging methods obscure detail in the reaction profile. Our results highlight the importance of explicit environmental models and suggest that asparagine N624 serves a secondary function of reducing spontaneous mutations in PcrA Helicase.

Quantum Tunnelling Effects in the Guanine-Thymine Wobble Misincorporation via Tautomerism.

The misincorporation of a noncomplementary DNA base in the polymerase active site is a critical source of replication errors that can lead to genetic mutations. In this work, we model the mechanism of wobble mispairing and the subsequent rate of misincorporation errors by coupling first-principles quantum chemistry calculations to an open quantum systems master equation. This methodology allows us to accurately calculate the proton transfer between bases, allowing the misincorporation and formation of mutagenic tautomeric forms of DNA bases. Our calculated rates of genetic error formation are in excellent agreement with experimental observations in DNA. Furthermore, our quantum mechanics/molecular mechanics model predicts the existence of a short-lived "tunnelling-ready" configuration along the wobble reaction pathway in the polymerase active site, dramatically increasing the rate of proton transfer by a hundredfold, demonstrating that quantum tunnelling plays a critical role in determining the transcription error frequency of the polymerase.

Tautomerisation Mechanisms in the Adenine-Thymine Nucleobase Pair during DNA Strand Separation.

The adenine-thymine tautomer (A*-T*) has previously been discounted as a spontaneous mutagenesis mechanism due to the energetic instability of the tautomeric configuration. We study the stability of A*-T* while the nucleobases undergo DNA strand separation. Our calculations indicate an increase in the stability of A*-T* as the DNA strands unzip and the hydrogen bonds between the bases stretch. Molecular Dynamics simulations reveal the time scales and dynamics of DNA strand separation and the statistical ensemble of opening angles present in a biological environment. Our results demonstrate that the unwinding of DNA, an inherently out-of-equilibrium process facilitated by helicase, will change the energy landscape of the adenine-thymine tautomerization reaction. We propose that DNA strand separation allows the stable tautomerization of adenine-thymine, providing a feasible pathway for genetic point mutations via proton transfer between the A-T bases.

Proton transfer during DNA strand separation as a source of mutagenic guanine-cytosine tautomers.

Proton transfer between the DNA bases can lead to mutagenic Guanine-Cytosine tautomers. Over the past several decades, a heated debate has emerged over the biological impact of tautomeric forms. Here, we determine that the energy required for generating tautomers radically changes during the separation of double-stranded DNA. Density Functional Theory calculations indicate that the double proton transfer in Guanine-Cytosine follows a sequential, step-like mechanism where the reaction barrier increases quasi-linearly with strand separation. These results point to increased stability of the tautomer when the DNA strands unzip as they enter the helicase, effectively trapping the tautomer population. In addition, molecular dynamics simulations indicate that the relevant strand separation time is two orders of magnitude quicker than previously thought. Our results demonstrate that the unwinding of DNA by the helicase could simultaneously slow the formation but significantly enhance the stability of tautomeric base pairs and provide a feasible pathway for spontaneous DNA mutations.