Autofluorescent Artemisinin-Benzimidazole Hybrids via Organo-Click Reaction: Study of Antiviral Properties and Mode of Action in Living Cells.

Drug modification by a fluorescent label is a common tool for studying its mechanism of action with fluorescence microscopy techniques. However, the attachment of a fluorescent label can significantly alter the polarity, solubility, and biological activity of the investigated drug, and, as a result, the studied mechanism of action can be misrepresented. Therefore, developing efficient drugs, which are inherently fluorescent and can be tracked directly in the cell is highly favorable. Here an easy formation of fluorescent hybrid drugs is presented, generated by a combination of two readily available non-fluorescent pharmacophores via a non-cleavable linker using a Ramachary-Bressy-Wang organocatalyzed azide-carbonyl [3+2] cycloaddition (organo-click) reaction. All newly prepared fluorescent compounds showed strong anti-HCMV activity (EC50 down to 0.07±0.00 µM), thus presenting a very promising drug developmental basis compared to the approved drug ganciclovir (EC50 2.60±0.50 µM). Remarkably, in vitro fluorescent imaging investigation of new compounds revealed induced changes in mitochondrial structures, which is a phenotypical hallmark of antiviral activity. This approach opens up new vistas for the easy formation of potent fluorescent drugs from readily available non-fluorescent parent compounds and might facilitate insight into their mode of action in living cells, avoiding the requirement of linkage to external fluorescent markers.

Anti-SARS-CoV-2 Inhibitory Profile of New Quinoline Compounds in Cell Culture-Based Infection Models.

Invited for the cover of this issue are Manfred Marschall, Svetlana B. Tsogoeva and co-workers at Friedrich-Alexander University of Erlangen-Nürnberg. The image depicts a new anti-SARS-CoV-2 compound in front of SARS-CoV-2 viruses. Read the full text of the article at 10.1002/chem.202103861.

Anti-SARS-CoV-2 Inhibitory Profile of New Quinoline Compounds in Cell Culture-Based Infection Models.

The presently ongoing pandemic of human SARS-CoV-2 infections (COVID-19) presents an enormous challenge in surveillance, vaccine and antiviral drug development. Here we report the synthesis of new bioactive quinoline-morpholine hybrid compounds and their virological evaluation, which proves pronounced cell culture-based inhibitory profile against SARS-CoV-2. Thus, selected quinoline compounds may suggest specific hit-to-lead development.

Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations.

Human cytomegalovirus (HCMV) is a pathogenic human herpesvirus associated with serious, potentially life-threatening symptoms in the immunocompromised or immunonaïve host. The limitations encountered by antiviral therapy options currently available include a narrow panel of accessible targets, the induction of viral drug resistance as well as severe drug dosage-mediated side-effects. Improved drug-targeting strategies to resolve these issues are the focus of our investigations. In particular, pharmaceutical kinase inhibitors (PKIs), either directed to host kinases or directed to the viral protein kinase pUL97, have been considered to overcome these restrictions. Recently, we reported the identification of a synergistic combination of two PKIs directed to host cyclin-dependent kinase 7 (CDK7) and viral CDK ortholog pUL97. Here, we substantiate these findings with the following results: (i) true drug synergy was exhibited by various chemical classes of PKI pairs directed to pUL97 and CDK7; (ii) no putative amplification of cytotoxicity by these drug combinations was observed; (iii) a reduction in drug dosage levels for synergistic combinations was defined on a quantitative basis and compared to monotreatments; (iv) the quantities of target proteins CDK7 and pUL97 expressed in HCMV-infected cells were assessed by confocal imaging, indicating a strong down-modulation of CDK7 levels as a result of synergistic drug treatment; (v) the functional importance of these target kinases, both binding to cyclin H, was illustrated by assessing HCMV replication under the viral genomic deletion of ORF-UL97 or cellular cyclin knock-out; (vi) new combinations of HCMV-specific drug synergy were demonstrated for solely host-directed treatments using PKIs against CDK2, CDK7, CDK8 and/or CDK9 and (vii) a triple PKI combination provided further support for the synergy approach. With these combined findings, this study highlights the potential of therapeutic drug combinations of approved, developmental and preclinical PKIs for expanding future options for anti-HCMV therapy.

Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs).

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, ß- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97.

Human cytomegalovirus (HCMV) is a common ß-herpesvirus causing life-long latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97-cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.

(Iso)Quinoline-Artemisinin Hybrids Prepared through Click Chemistry: Highly Potent Agents against Viruses.

Viral infections cause life-threatening diseases in millions of people worldwide every year and there is an urgent need for new, effective antiviral drugs. Hybridization of two chemically diverse compounds into a new bioactive effector product is a successful concept to improve the properties of a hybrid drug relative to the parent compounds. In this study, (iso)quinoline-artemisinin hybrids, obtained through copper-catalyzed azide-alkyne cycloaddition or metal-free click reactions (in organic solvents or in the presence of water), were analyzed in vitro, for the first time, for their inhibitory activity against human cytomegalovirus (HCMV), relative to their parent compounds and the reference drug ganciclovir. EC50 (HCMV) values were obtained in a range 0.22-1.20 µm, which indicated highly potent antiviral properties in the absence of cytotoxic effects on normal cells (CC50 >100 µm). The most active hybrid, 1 (EC50 =0.22 µm), is 25 times more potent than its parent compound artesunic acid (EC50 =5.41 µm) and 12 times more efficient than the standard drug ganciclovir (EC50 =2.6 µm). Interestingly, hybrid 1 also shows inhibitory activity against hepatitis B virus in vitro (EC50 (HBeAg)=2.57 µm).

The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism.

Human cytomegalovirus (HCMV) is a major human pathogen associated with severe pathology. Current options of antiviral therapy only partly satisfy the needs of a well-tolerated long-term treatment/prophylaxis free from drug-induced viral resistance. Recently, we reported the strong antiviral properties in vitro and in vivo of the broad-spectrum anti-infective drug artesunate and its optimized derivatives. NF-κB signaling was described as a targeting mechanism and additional target proteins have recently been identified. Here, we analyzed the autofluorescent hybrid compound BG95, which could be utilized for intracellular visualization by confocal imaging and a tracking analysis in virus-infected primary human fibroblasts. As an important finding, BG95 accumulated in mitochondria visualized by anti-prohibitin and MitoTracker staining, and induced statistically significant changes of mitochondrial morphology, distinct from those induced by HCMV infection. Notably, mitochondrial membrane potential was found substantially reduced by BG95, an effect apparently counteracting efficient HCMV replication, which requires active mitochondria and upregulated energy levels. This finding was consistent with binding properties of artesunate-like compounds to mitochondrial proteins and thereby suggested a new mechanistic aspect. Combined, the present study underlines an important role of mitochondria in the multifaceted, host-directed antiviral mechanism of this drug class, postulating a new mitochondria-specific mode of protein targeting.

Visible-Light-Driven C-H Oxidation of Cyclic Tertiary Amines: Access to Synthetic Strychnos Alkaloids with Antiviral Activity.

Air and visible light have been used in facile direct C-H oxidation of cyclic tertiary amines at ambient conditions, employing organic dyes as photocatalysts and LED. Tolerance of this new environmentally compatible protocol to various side-chain derivatizations of tryptoline and tetrahydroisoquinoline substrates was demonstrated. The developed method provides a straightforward and sustainable route towards δ-lactams, which feature strong antiviral properties (EC50 down to 4.6±1.8 µm) against human cytomegalovirus (HCMV). The clear advantages, which are easily available and inexpensive reagents, organic dyes, visible light, air/O2 and atom efficiency, make this system highly appealing for synthesis of versatile Strychnocarpine alkaloid derivatives with antiviral activity.

Synthesis of new betulinic acid/betulin-derived dimers and hybrids with potent antimalarial and antiviral activities.

Severe malaria and viral infections cause life-threatening diseases in millions of people worldwide every year. In search for effective bioactive hybrid molecules, which may possess improved properties compared to their parent compounds, a series of betulinic acid/betulin based dimer and hybrid compounds carrying ferrocene and/or artesunic acid moieties, was designed and, synthesized de novo. Furthermore, they were analyzed in vitro against malaria parasites (growth inhibition of 3D7-strain P. falciparum-infected erythrocytes) and human cytomegalovirus (HCMV). From this series of hybrids/dimers, the betulinic acid/betulin and artesunic acid hybrids 11 and 12 showed the most potent activities against P. falciparum and HCMV. On the strength of results, additive and/or synergistic effects between the natural or semisynthetic products, such as betulinic acid-/betulin- and artesunic acid-derived compounds, are suggested on the basis of putatively complex modes of antimicrobial action. This advantage may be taken into account in future drug development.

Synthesis of Artemisinin-Derived Dimers, Trimers and Dendrimers: Investigation of Their Antimalarial and Antiviral Activities Including Putative Mechanisms of Action.

Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and investigate their action against malaria parasite Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV). Dimer 7 was the most active compound (EC50 1.4 nm) in terms of antimalarial efficacy and was even more effective than the standard drugs dihydroartemisinin (EC50 2.4 nm), artesunic acid (EC50 8.9 nm) and chloroquine (EC50 9.8 nm). Trimer 4 stood out as the most active agent against HCMV in vitro replication and exerted an EC50 value of 0.026 µm, representing an even higher activity than the two reference drugs ganciclovir (EC50 2.60 µm) and artesunic acid (EC50 5.41 µm). In addition, artemisinin-derived dimer 13 and trimer 15 were for the first time both immobilized on TOYOPEARL AF-Amino-650M beads and used for mass spectrometry-based target identification experiments using total lysates of HCMV-infected primary human fibroblasts. Two major groups of novel target candidates, namely cytoskeletal and mitochondrial proteins were obtained. Two putatively compound-binding viral proteins, namely major capsid protein (MCP) and envelope glycoprotein pUL132, which are both essential for HCMV replication, were identified.

Access to new highly potent antileukemia, antiviral and antimalarial agents via hybridization of natural products (homo)egonol, thymoquinone and artemisinin.

Hybridization of natural products has high potential to further improve their activities and may produce synergistic effects between linked pharmacophores. Here we report synthesis of nine new hybrids of natural products egonol, homoegonol, thymoquinone and artemisinin and evaluation of their activities against P. falciparum 3D7 parasites, human cytomegalovirus, sensitive and multidrug-resistant human leukemia cells. Most of the new hybrids exceed their parent compounds in antimalarial, antiviral and antileukemia activities and in some cases show higher in vitro efficacy than clinically used reference drugs chloroquine, ganciclovir and doxorubicin. Combined, our findings stress the high potency of these hybrids and encourages further use of the hybridization concept in applied pharmacological research.

HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein.

The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p6(23-52) S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.

Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30-70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of ß-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66-71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix-loop-helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.

Preliminary study on carprofen concentration measurements after transcutaneous treatment with Vetdrop® in a microfracture joint defect model in sheep.

BACKGROUND: The present preliminary study describes concentration time courses of the NSAID carprofen in the plasma and synovial fluid in a microfrature sheep model after transcutaneous treatments with a novel application device (Vetdrop®). To treat circumscribed inflammatory processes a transcutaneous application device could potentially be beneficial. After transcutaneous application normally lower systemic concentrations are measured which may reduce the incidence of side effects, whereas efficacy is still maintained. In this study carprofen was used based on its capacity to provide analgesia after orthopaedic procedures in sheep and it is considered that it may have a positive influence on the healing of cartilage in low concentrations. RESULTS: In all transcutaneously treated animals, carprofen plasma concentrations exceeded those of synovial fluid, although plasma levels remained significantly reduced (300-fold) as compared to carprofen administered intravenously. Furthermore, in contrast to the intravenously treated animals, a modest accumulation of carprofen in plasma and synovial fluid was observed in the transcutaneously treated animals over the 6-week treatment period. CONCLUSIONS: The transcutaneously administered carprofen using the Vetdrop® device penetrated the skin and both, plasma- and synovial concentrations could be measured repeatedly over time. This novel device may be considered a valuable transcutaneous drug delivery system.

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels.

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORß, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

Synthesis and Characterization of DHODH Inhibitors Based on the Vidofludimus Scaffold with Pronounced Anti-SARS-CoV-2 Activity.

New strategies for the rapid development of broad-spectrum antiviral therapies are urgently required for emerging and re-emerging viruses like the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Host-directed antivirals that target universal cellular metabolic pathways necessary for viral replication present a promising approach with broad-spectrum activity and low potential for development of viral resistance. Dihydroorotate dehydrogenase (DHODH) was identified as one of those universal host factors essential for the replication of many clinically relevant human pathogenic viruses. DHODH is the rate-limiting enzyme catalyzing the fourth step in the de novo pyrimidine synthesis. Therefore, it is also developed as a therapeutic target for many diseases relying on cellular pyrimidine resources, such as cancer, autoimmune diseases and viral or bacterial infection. Thus, several DHODH inhibitors, including vidofludimus calcium (VidoCa, IMU-838), are currently in development or have been investigated in clinical trials for the treatment of virus infections such as SARS-CoV-2-mediated coronavirus disease 19 (COVID-19). Here, we report the medicinal chemistry optimization of VidoCa that resulted in metabolically more stable derivatives with improved DHODH target inhibition in various mammalian species, which translated into improved efficacy against SARS-CoV-2.

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target.

Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.