C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Synthesis and evaluation of 3-alkynyl-5-aryl-7-aza-indoles as broad-spectrum antiviral agents.

RNA viral infections, including those caused by respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Venezuelan Equine encephalitis virus (VEEV), pose a major global health challenge. Here, we report the synthesis and screening of a series of pyrrolo[2,3-b]pyridines targeting RSV, SARS-CoV-2 and/or VEEV. From this campaign, a series of lead compounds was generated that demonstrated antiviral activity in the low single-digit micromolar range against the various viruses and did not show cytotoxicity. These findings highlight the potential of 3-alkynyl-5-aryl-7-aza-indoles as a promising chemotype for the development of broad-spectrum antiviral agents.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

Organotropic dendrons with high potency as HIV-1, HIV-2 and EV-A71 cell entry inhibitors.

We have recently described a novel family of compounds of reduced size and dual anti-HIV and anti-EV71 activity that encompasses tripodal and tetrapodal derivatives. The tripodal prototype, AL-470, has a nitro group at the focal point of the central scaffold and three attached tryptophan residues, each of which bearing an isophthaloyl moiety at the C2 position of the indole ring. A nitro to amino substitution has allowed us now to introduce a chemically addressable functionality to perform further structural modifications consisting of both direct and linker-mediated attachment of several aromatic groups, including the fluorescent dye Alexa Fluor 647 and the antibody-recruiting 2,4-dinitrophenyl motif. Some of the derivatives turned out to be more potent and selective than AL-470 against HIV-1, HIV-2 and EV-A71. The fluorescent probe demonstrated a specific tropism for intestines and lungs, two important niches for the human microbiome in health and disease.

Double Arylation of the Indole Side Chain of Tri- and Tetrapodal Tryptophan Derivatives Renders Highly Potent HIV-1 and EV-A71 Entry Inhibitors†.

We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.

A patent review of adaptor associated kinase 1 (AAK1) inhibitors (2013-present).

INTRODUCTION: Adaptor-associated kinase 1 (AAK1) has been proposed as being a promising drug target for the treatment of a variety of neurological and psychiatric disorders, such as schizophrenia, cognitive deficits in schizophrenia, Parkinson's disease, bipolar disorder, Alzheimer's disease and neuropathic pain. More recently, AAK1 was shown to be an essential cellular factor for viral replication and therefore has been pursued as a host target for the development of broad-spectrum antiviral agents. AREAS COVERED: This review provides an overview of the patented AAK1 inhibitors from 2013 to present. EXPERT OPINION: The promise of AAK1 as drug target for the treatment of neuropathic pain stimulated the search for AAK1 inhibitors. However, only two companies (i.e. Lexicon Pharmaceuticals and Bristol Myers Squibb) seemed to be active in this field and filed patent applications in the last few years. The most promising congeners showed promising in vitro activity in a variety of AAK1-related assays. Moreover, selected compounds were also endowed with in vivo activity in various preclinical animal models for neuropathic pain.

Discovery of 3-phenyl- and 3-N-piperidinyl-isothiazolo[4,3-b]pyridines as highly potent inhibitors of cyclin G-associated kinase.

Structural modifications at position 3 of the isothiazolo[4,3-b]pyridine scaffold afforded a new series of cyclin G-associated kinase (GAK) inhibitors. It was shown that the insertion of a carboxamide residue at position 3 of a phenyl or piperidinyl moiety generated potent GAK inhibitors with IC50 values in a low nanomolar range. This potent GAK binding affinity was rationalized by molecular modelling demonstrating that the carboxamide moiety engages in an extra hydrogen bond with GAK. Moreover, this new series of compounds was also endowed with antiviral activity against dengue virus, highlighting the potential utility of GAK as a target for the development of antiviral drugs.

Tryptophan Trimers and Tetramers Inhibit Dengue and Zika Virus Replication by Interfering with Viral Attachment Processes.

Here, we report a class of tryptophan trimers and tetramers that inhibit (at low micromolar range) dengue and Zika virus infection in vitro These compounds (AL family) have three or four peripheral tryptophan moieties directly linked to a central scaffold through their amino groups; thus, their carboxylic acid groups are free and exposed to the periphery. Structure-activity relationship (SAR) studies demonstrated that the presence of extra phenyl rings with substituents other than COOH at the N1 or C2 position of the indole side chain is a requisite for the antiviral activity against both viruses. The molecules showed potent antiviral activity, with low cytotoxicity, when evaluated on different cell lines. Moreover, they were active against laboratory and clinical strains of all four serotypes of dengue virus as well as a selected group of Zika virus strains. Additional mechanistic studies performed with the two most potent compounds (AL439 and AL440) demonstrated an interaction with the viral envelope glycoprotein (domain III) of dengue 2 virus, preventing virus attachment to the host cell membrane. Since no antiviral agent is approved at the moment against these two flaviviruses, further pharmacokinetic studies with these molecules are needed for their development as future therapeutic/prophylactic drugs.

Structure-activity relationship study of the pyridine moiety of isothiazolo[4,3-b]pyridines as antiviral agents targeting cyclin G-associated kinase.

Previously, we reported the discovery of 3,6-disubstituted isothiazolo[4,3-b]pyridines as potent and selective cyclin G-associated kinase (GAK) inhibitors with promising antiviral activity. In this manuscript, the structure-activity relationship study was expanded to synthesis of isothiazolo[4,3-b]pyridines with modifications of the pyridine moiety. This effort led to the discovery of an isothiazolo[4,3-b]pyridine derivative with a 3,4-dimethoxyphenyl residue at position 5 that displayed low nanomolar GAK binding affinity and antiviral activity against dengue virus.

Scaffold Simplification Strategy Leads to a Novel Generation of Dual Human Immunodeficiency Virus and Enterovirus-A71 Entry Inhibitors.

Currently, there are only three FDA-approved drugs that inhibit human immunodeficiency virus (HIV) entry-fusion into host cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71. These compounds contain in their structure three or four tryptophan (Trp) residues linked to a central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings, bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and the fivefold axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis, and great opportunities for chemical optimization make them useful prototypes for future medicinal chemistry studies.

Modifications in the branched arms of a class of dual inhibitors of HIV and EV71 replication expand their antiviral spectrum.

We have previously reported a new class of dendrimers with tryptophan (Trp) residues on the surface that show dual antiviral activities against HIV and enterovirus EV71. The prototype compound of this family is a derivative of pentaerythritol with 12 peripheral Trp groups and trivalent spacer arms. Here a novel series of dendrimers with divalent and tetravalent branched arms, instead of the trivalent ones present on the prototype, has been synthesized and its activity against HIV, EV71 and a panel of 16 different viruses and other pathogens has been determined. Convergent or divergent approaches have been used for the synthesis of these compounds. Our findings demonstrate that only compounds with tetravalent branched arms showed the same anti-HIV and anti-EV71 activity of the prototype (low micromolar) and even gain significant antiviral activity against new pathogens such as HSV-2, adenovirus-2, human corona virus and respiratory syncytial virus, being the first members of the Trp dendrimer family that showed activity against those viruses. As the prototype, these compounds also showed low-nanomolar activity against a representative EV71 clinical isolate. Experimental work carried on to determine the mode of action of the most potent IIa, containing tetravalent branched arms, demonstrated that it interacts with the viral envelopes of HIV, EV71 and HSV-2 and thus may prevent virus attachment to the host cell. These results support the interest of this new series of Trp dendrimers and qualify them as useful prototypes for the development of novel inhibitors of viral entry with broad antiviral spectrum.

Viral engagement with host receptors blocked by a novel class of tryptophan dendrimers that targets the 5-fold-axis of the enterovirus-A71 capsid.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Galloyl Carbohydrates with Antiangiogenic Activity Mediated by Capillary Morphogenesis Gene 2 (CMG2) Protein Binding.

We previously showed that a small molecule of natural origin, 1,2,3,4,6-penta- O-galloyl-ß-d-glucopyranose (PGG), binds to capillary morphogenesis gene 2 (CMG2) with a submicromolar IC50 and also has antiangiogenic activity in vitro and in vivo. In this work, we synthetized derivatives of PGG with different sugar cores and phenolic substituents and tested these as angiogenesis inhibitors. In a high-throughput Förster resonant energy transfer-based binding assay, we found that one of our synthetic analogues (1,2,3,4,6-penta- O-galloyl-ß-d-mannopyranose (PGM)), with mannose as central core and galloyl substituents, exhibit higher (up to 10×) affinity for CMG2 than the natural glucose prototype PGG and proved to be a potent angiogenesis inhibitor. These findings demonstrate that biochemical CMG2 binding in vitro predicts inhibition of endothelial cell migration ex vivo and antiangiogenic activity in vivo. The molecules herein described, and in particular PGM, might be useful prototypes for the development of novel agents for angiogenesis-dependent diseases, including blinding eye disease and cancer.

Structure-activity relationship studies on a Trp dendrimer with dual activities against HIV and enterovirus A71. Modifications on the amino acid.

We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship studies of this family to identify key features that might be significant for the antiviral activity. With this aim, novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a "decarboxylated" analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71 clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the divergent approach previously reported.

Optimization of a Class of Tryptophan Dendrimers That Inhibit HIV Replication Leads to a Selective, Specific, and Low-Nanomolar Inhibitor of Clinical Isolates of Enterovirus A71.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range.