Citations

We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN’s toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10–20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).

Abstract Coronavirus disease 2019 (COVID-19) infection is associated with risk of persistent neurocognitive and neuropsychiatric complications, termed “long COVID”. It is unclear whether the neuropsychological manifestations of COVID-19 present as a uniform syndrome or as distinct neurophenotypes with differing risk factors and recovery outcomes. We examined post-acute neuropsychological profiles following SARS-CoV-2 infection in 205 patients recruited from inpatient and outpatient populations, using an unsupervised machine learning cluster analysis, with objective and subjective measures as input features. This resulted in three distinct post-COVID clusters. In the largest cluster (69%), cognitive functions were within normal limits, although mild subjective attention and memory complaints were reported. Vaccination was associated with membership in this “normal cognition” phenotype. Cognitive impairment was present in the remaining 31% of the sample but clustered into two differentially impaired groups. In 16% of participants, memory deficits, slowed processing speed, and fatigue were predominant. Risk factors for membership in the “memory-speed impaired” neurophenotype included anosmia and more severe COVID-19 infection. In the remaining 15% of participants, executive dysfunction was predominant. Risk factors for membership in this milder “dysexecutive” neurophenotype included disease-nonspecific factors such as neighborhood deprivation and obesity. Recovery outcomes at 6-month follow-up differed across neurophenotypes, with the normal cognition group showing improvement in verbal memory and psychomotor speed, the dysexecutive group showing improvement in cognitive flexibility, and the memory-speed impaired group showing no objective improvement and relatively worse functional outcomes compared to the other two clusters. These results indicate that there are multiple post-acute neurophenotypes of long COVID, with different etiological pathways and recovery outcomes. This information may inform phenotype-specific approaches to treatment.

Abstract Coronavirus disease 2019 (COVID-19) infection is associated with risk of persistent neurocognitive and neuropsychiatric complications, termed “long COVID”. It is unclear whether the neuropsychological manifestations of COVID-19 present as a uniform syndrome or as distinct neurophenotypes with differing risk factors and recovery trajectories. We examined post-acute outcomes following SARS-CoV-2 infection in 205 patients recruited from inpatient and outpatient populations, using an unsupervised machine learning cluster analysis, with objective and subjective neuropsychological measures as input features. This resulted in three distinct post-COVID clusters. In the largest cluster (69%), cognitive functions were within normal limits (“normal cognition” neurophenotype), although mild subjective attention and memory complaints were reported. Cognitive impairment was present in the remaining 31% of the sample but clustered into two differentially impaired groups. In 16% of participants, memory deficits, slowed processed speed, and fatigue were predominant. Risk factors for membership in the “memory-speed impaired” neurophenotype included anosmia and more severe COVID-19 infection. In the remaining 15% of participants, executive dysfunction was predominant. Risk factors for membership in this milder “dysexecutive” neurophenotype included disease-nonspecific factors such as neighborhood deprivation and obesity. Recovery trajectories at 6-month follow-up differed across neurophenotypes, with the normal cognition group showing stability, the dysexecutive group showing improvement, and the memory-speed impaired group showing persistent processing speed deficits and fatigue, as well as worse functional outcomes. These results indicate that there are multiple post-acute neurophenotypes of long COVID, with different etiological pathways and recovery trajectories. This information may inform phenotype-specific approaches to treatment.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, ‘Calxinin’. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Leishmaniasis remains a serious public health problem in many tropical regions of the world. Among neglected tropical diseases, the mortality rate of leishmaniasis is second only to malaria. All currently approved therapeutics have toxic side effects and face rapidly increasing resistance. To identify existing drugs with antileishmanial activity and predict the mechanism of action, we designed a drug-discovery pipeline utilizing both in-silico and in-vitro methods. First, we screened compounds from the Selleckchem Bio-Active Compound Library containing ~1,622 FDA-approved drugs and narrowed these down to 96 candidates based on data mining for possible anti-parasitic properties. Next, we completed preliminary in-vitro testing of compounds against Leishmania amastigotes and selected the most promising active compounds, Lansoprazole and Posaconazole. We identified possible Leishmania drug targets of Lansoprazole and Posaconazole using several available servers. Our in-silico screen identified likely Lansoprazole targets as the closely related calcium-transporting ATPases (LdBPK_352080.1, LdBPK_040010.1, and LdBPK_170660.1), and the Posaconazole target as lanosterol 14-alpha-demethylase (LdBPK_111100.1). Further validation showed LdBPK_352080.1 to be the most plausible target based on induced-fit docking followed by long (100ns) MD simulations to confirm the stability of the docked complexes. We present a likely ion channel-based mechanism of action of Lansoprazole against Leishmania calcium-transporting ATPases, which are essential for parasite metabolism and infectivity. The LdBPK_111100.1 interaction with Posaconazole is very similar to the known fungal orthologue. Herein, we present two novel anti-leishmanial agents, Posaconazole and Lansoprazole, already approved by the FDA for different indications and propose plausible mechanisms of action for their antileishmanial activity.

Abstract After more than a year of the COVID‐19 pandemic, SARS‐CoV‐2 infection rates with newer variants continue to devastate much of the world. Global healthcare systems are overwhelmed with high positive patient numbers. Silent hypoxia accompanied by rapid deterioration and some cases with septic shock is responsible for COVID‐19 mortality in many hospitalized patients. There is an urgent need to further understand the relationships and interplay with human host components during pathogenesis and immune evasion strategies. Currently, acquired immunity through vaccination or prior infection usually provides sufficient protection against the emerging variants of SARS‐CoV‐2 except Omicron variant requiring recent booster. New strains have shown higher viral loads and greater transmissibility with more severe disease presentations. Notably, COVID‐19 has a peculiar prognosis in severe patients with iron dysregulation and hypoxia which is still poorly understood. Studies have shown abnormally low serum iron levels in severe infection but a high iron overload in lung fibrotic tissue. Data from our in‐silico structural analysis of the spike protein sequence along with host proteolysis processing suggests that the viral spike protein fragment mimics Hepcidin and is resistant to the major human proteases. This functional spike‐derived peptide dubbed “Covidin” thus may be intricately involved with host ferroportin binding and internalization leading to dysregulated host iron metabolism. Here, we propose the possible role of this potentially allogenic mimetic hormone corresponding to severe COVID‐19 immunopathology and illustrate that this molecular mimicry is responsible for a major pathway associated with severe disease status. Furthermore, through 3D molecular modeling and docking followed by MD simulation validation, we have unraveled the likely role of Covidin in iron dysregulation in COVID‐19 patients. Our meta‐analysis suggests the Hepcidin mimetic mechanism is highly conserved among its host range as well as among all new variants to date including Omicron. Extensive analysis of current mutations revealed that new variants are becoming alarmingly more resistant to selective human proteases associated with host defense.

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

A novel hydroxyethylamine compound was synthesised that displayed biological potency against SARS-CoV-2.

AbstractCalcium channels (CCs), a group of ubiquitously expressed membrane proteins, are involved in many pathophysiological processes of protozoan parasites. Our understanding of CCs in cell signaling, organelle function, cellular homeostasis, and cell cycle control has led to improved insights into their structure and functions. In this article, we discuss CCs characteristics of five major protozoan parasites Plasmodium, Leishmania, Toxoplasma, Trypanosoma, and Cryptosporidium. We provide a comprehensive review of current antiparasitic drugs and the potential of using CCs as new therapeutic targets. Interestingly, previous studies have demonstrated that human CC modulators can kill or sensitize parasites to antiparasitic drugs. Still, none of the parasite CCs, pumps, or transporters has been validated as drug targets. Information for this review draws from extensive data mining of genome sequences, chemical library screenings, and drug design studies. Parasitic resistance to currently approved therapeutics is a serious and emerging threat to both disease control and management efforts. In this article, we suggest that the disruption of calcium homeostasis may be an effective approach to develop new anti‐parasite drug candidates and reduce parasite resistance.