Dense tumor-infiltrating lymphocytes (TILs) in liver metastasis from colorectal cancer are related to improved overall survival.

BACKGROUND AND OBJECTIVES: Tumor-infiltrating lymphocytes (TILs) represent a host-tumor interaction, frequently signifying an augmented immunological response. Nonetheless, implications with survival outcomes in patients with colorectal carcinoma liver metastasis (CRLM) warrant rigorous validation. The objective was to demonstrate the association between TILs and survival in patients with CRLM. METHOD: In a retrospective evaluation conducted in a single institution, we assessed all patients who underwent hepatectomy due to CRLM between 2014 and 2018. Comprehensive medical documentation reviews were executed. TILs were assessed by a liver pathologist, blinded to the clinical information, in all surgical slides. RESULTS: This retrospective cohort included 112 patients. Median overall survival (OS) was 58 months and disease-free survival (DFS) was 12 months for the entire cohort. Comparison between groups showed a median OS of 81 months in the dense TILs group and 40 months in the weak/absent group (p = 0.001), and DFS was 14 months versus 9 months (p = 0.041). Multivariable analysis showed that TILs were an independent predictor of OS (HR 1.95; p = 0.031). CONCLUSIONS: Dense TILs are a pivotal prognostic indicator, correlating with enhanced OS. Including TILs information in histopathological evaluations should refine the clinical decision-making process for this group of patients.

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.

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.

Discovery of PLpro and Mpro Inhibitors for SARS-CoV-2.

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (Mpro) and the papain-like protease (PLpro) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against Mpro and PLpro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for Mpro and 8 hits for PLpro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC50 = 2.72 ± 0.09 µM for PLpro and IC50 = 7.25 ± 0.15 µM for Mpro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene (IC50 = 3.28 ± 0.29 µM for PLpro and IC50 = 42.8 ± 6.7 µM for Mpro). We additionally tested several kinase inhibitors and identified olmutinib (IC50 = 0.54 ± 0.04 µM), bosutinib (IC50 = 4.23 ± 0.28 µM), crizotinib (IC50 = 3.81 ± 0.04 µM), and dacominitinib (IC50 = IC50 3.33 ± 0.06 µM) as PLpro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PLpro may provide new repurposing opportunities or starting points for chemical optimization.

Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.

The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.

Structural basis of nirmatrelvir and ensitrelvir activity against naturally occurring polymorphisms of the SARS-CoV-2 main protease.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.

Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways.

The Chikungunya virus (CHIKV) causes Chikungunya fever, a disease characterized by symptoms such as arthralgia/polyarthralgia. Currently, there are no antivirals approved against CHIKV, emphasizing the need to develop novel therapies. The imidazonaphthyridine compound (RO8191), an interferon-α (IFN-α) agonist, was reported as a potent inhibitor of HCV. Here RO8191 was investigated for its potential to inhibit CHIKV replication in vitro. RO8191 inhibited CHIKV infection in BHK-21 and Vero-E6 cells with a selectivity index (SI) of 12.3 and 37.3, respectively. Additionally, RO8191 was capable to protect cells against CHIKV infection, inhibit entry by virucidal activity, and strongly impair post-entry steps of viral replication. An effect of RO8191 on CHIKV replication was demonstrated in BHK-21 through type-1 IFN production mechanism and in Vero-E6 cells which has a defective type-1 IFN production, also suggesting a type-1 IFN independent mode of action. Molecular docking calculations demonstrated interactions of RO8191 with the CHIKV E proteins, corroborated by the ATR-FTIR assay, and with non-structural proteins, supported by the CHIKV-subgenomic replicon cells assay.

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

Surgery or active surveillance for pNETs < 2 cm: Preliminary results from a single center Brazilian cohort.

BACKGROUND AND OBJECTIVES: Incidence of pancreatic neuroendocrine tumors (pNETS) seems to be rising over the years, with many cases incidentally diagnosed. Surgery and active surveillance are current treatment modalities for small pNETS. We review our institutional series and compare outcomes for small asymptomatic and nonfunctioning tumors. METHODS: This retrospective cohort study included patients with 2 cm or less and well differentiated pNETS at a single Brazilian Cancer Center. From 2002 to 2020, patients received active surveillance or surgery as a treatment strategy. Short and long-term results were compared. RESULTS: Sixty-four patients were included, 41 in surgical strategy and 23 in the active surveillance approach. Baseline group characteristics were comparable. More patients on active surveillance underwent abdominal magnetic resonance imaging (MRI) and had tumors located in the pancreatic head (41% vs. 17%, p = 0.038). Minimally invasive procedure was chosen in 80.1% of the surgical patients. No patient died after surgery. Median follow-up period was 38.6 and 46.4 months for active surveillance and surgery cohorts, respectively. No difference in disease progression rate was observed. CONCLUSION: Both approaches seem to be safe for small pNETs. Long-term outcome and quality of life should be considered when discussing such options with patients.

PD-L1 expression in gastric and gastroesophageal junction cancer patients treated with perioperative chemotherapy.

BACKGROUND AND OBJECTIVES: The incidence, predictive, and prognostic impact of programmed cell death (PD-L1) expression in gastric (GC) and gastroesophageal junction tumors (GEJC) treated with perioperative chemotherapy is poorly understood. We aimed to assess PD-L1 expression by immunohistochemistry (IHC) in both pre and posttreatment specimens evaluating its impact on pathological response and survival outcomes. METHODS: Retrospective cohort of patients with GC and GEJ tumors treated in a single western cancer center between 2007 and 2017. PD-L1 expression was assessed by IHC before and after neoadjuvant chemotherapy, in surgical samples, and reported as combined positive score (CPS). CPS > 1% was tested for its association with pathological response and overall survival (OS). RESULTS: We were able to assess PD-L1 expression in at least one tissue sample from 155 subjects. PD-L1 positivity rate was 20%. In 74 paired samples, a 21% discordance between PD-L1 expression in biopsy sample and surgical specimen was observed. With a median follow-up period of 60.3 months, 5-years disease-free survival was 60.5% with a median OS not reached. PD-L1 expression was neither associated with pathological response or survival outcomes. CONCLUSIONS: PD-L1 expression in the setting of locally advanced GC tumors was relatively low and can vary considering the tissue sample analyzed. This expression had no association with survival or pathological response in this population.

Characterization of the RNA-dependent RNA polymerase from Chikungunya virus and discovery of a novel ligand as a potential drug candidate.

Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

Patterns of disease relapse and posttreatment follow-up of patients with resected pancreatic adenocarcinoma: A single-center analysis.

BACKGROUND AND OBJECTIVES: To describe the patterns of disease relapse and follow-up of patients with resected pancreatic adenocarcinoma. Additionally, we looked at patients' characteristics at relapse and survival. METHODS: We included patients with potentially resectable pancreatic adenocarcinoma diagnosed from 2008 to 2018 who were submitted to resection with clear macroscopic margins and started posttreatment surveillance. RESULTS: The study population consists of 73 patients. The median interval between imaging studies was 3.2 months during the first 2 years of follow-up and 5.1 months thereafter. Forty-eight patients (65.8%) experienced disease relapse. The most frequent single site of relapse was locoregional (N = 21; 43.8%). At relapse, 31 patients (64.6%) were symptomatic and forty-two patients (87.6%) had Eastern Cooperative Oncology Group performance status 0 or 1. Most patients were able to undergo additional anticancer therapy (N = 41; 85.4%). Patients with asymptomatic relapses experienced longer median postrelapse survival (25.4 vs. 11.3 months; p = 0.015). CONCLUSIONS: A follow-up protocol that included imaging studies every 3 months in the first 2 years and every 6 months thereafter is able to diagnose disease relapse when patients have adequate performance status and are still able to undergo additional anticancer treatment.

Pyronaridine Protects against SARS-CoV-2 Infection in Mouse.

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency-approved for use against severe acute respiratory coronavirus 2 (SARS-CoV-2). One of these is remdesivir, which was originally repurposed from its use against Ebola. We evaluated three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg and identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. The in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS-CoV-2-infected mice, reducing lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 µM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

Symptomatic Recurrence and Survival Outcomes After Curative Treatment of Gastric Cancer: Does Intensive Follow-up Evaluation Improve Survival?

BACKGROUND: Intensive surveillance after treatment of gastric cancer patients with curative intent may lead to an earlier diagnosis of disease recurrence, but its impact on survival is uncertain. This study aimed to evaluate whether early diagnosis of disease recurrence among asymptomatic patients was associated with long-term survival. METHODS: This retrospective study analyzed patients with stages 1 to 3C gastric adenocarcinoma treated between 1999 and 2018. All recurrence events were classified as symptomatic or asymptomatic (detected by follow-up tests), and their clinicopathologic characteristics, patterns of recurrence, and survival were analyzed. RESULTS: The cohort consisted of 669 patients treated with a total gastrectomy in 48.6% and a D2-lymphadenectomy in 88.8% of the cases. Most of the tumors were pT3-4 (46.5%), with 45.5% involving lymph node metastases and 42.3% manifesting a diffuse histology. During a median follow-up period of 80.1 months (95% confidence interval [CI], 75.3-84.8 months), 166 patients had recurrences (24.8%), 65.7% of which were symptomatic. The peritoneum was the main site of recurrence (37.2%), and peritoneal recurrence was associated with worse overall survival (OS) (hazard ratio, 1.69; 95%CI, 1.2-2.37). The median disease-free, post-recurrence survival, and OS periods in the asymptomatic and symptomatic groups were respectively 13.4 versus 17.2 months (p = 0.04), 11.9 versus 4.7 months (p < 0.001), and 29.9 versus 26.4 months (p = 0.21). When OS was analyzed among the patients with non-peritoneal recurrence, no difference was observed between the two groups (31.3 vs 31.1 months; p = 0.46). CONCLUSION: Early diagnosis of asymptomatic disease recurrence did not affect the OS of the gastric cancer patients treated with curative intent. The use of intensive surveillance strategies in this scenario still requires further evidence.

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3'UTR-U30 (5'-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3').

Machine Learning Models Identify Inhibitors of SARS-CoV-2.

With the rapidly evolving SARS-CoV-2 variants of concern, there is an urgent need for the discovery of further treatments for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need, and numerous compounds have already been selected for in vitro testing by several groups. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein, we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA-approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, lumefantrine, an antimalarial was selected for testing and showed limited antiviral activity in cell-based assays while demonstrating binding (Kd 259 nM) to the spike protein using microscale thermophoresis. Several other compounds which we prioritized have since been tested by others and were also found to be active in vitro. This combined machine learning and in vitro testing approach can be expanded to virtually screen available molecules with predicted activity against SARS-CoV-2 reference WIV04 strain and circulating variants of concern. In the process of this work, we have created multiple iterations of machine learning models that can be used as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 with over 500 compounds is now freely available at www.assaycentral.org.