Understanding structure activity relationships of Good HEPES lipids for lipid nanoparticle mRNA vaccine applications.

Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.

Polygodial and Ophiobolin A Analogues for Covalent Crosslinking of Anticancer Targets.

In a search of small molecules active against apoptosis-resistant cancer cells, including glioma, melanoma, and non-small cell lung cancer, we previously prepared α,ß- and γ,δ-unsaturated ester analogues of polygodial and ophiobolin A, compounds capable of pyrrolylation of primary amines and demonstrating double-digit micromolar antiproliferative potencies in cancer cells. In the current work, we synthesized dimeric and trimeric variants of such compounds in an effort to discover compounds that could crosslink biological primary amine containing targets. We showed that such compounds retain the pyrrolylation ability and possess enhanced single-digit micromolar potencies toward apoptosis-resistant cancer cells. Target identification studies of these interesting compounds are underway.

Engaging Informal Private Health Care Providers for TB Case Detection: Experiences from RIPEND Project in India.

BACKGROUND: Informal (unqualified) health care providers are an important source of medical care for persons with presumptive TB (PPTB) in India. A project (titled RIPEND) was implemented to engage informal providers for the identification of PPTBs and TB patients in 4 districts of Telangana State, India, during October 2018-December 2019 project period. Engagement involved sensitizing the informal providers about TB, providing them financial incentives to identify PPTBs, and linking these PPTBs to diagnostic and treatment services provided by the Government of India's National TB Elimination Programme. OBJECTIVES: To describe (a) the characteristics of the informal providers, along with their self-reported practices on TB diagnosis, treatment, and challenges encountered by the RIPEND project staff in engaging them in the project and (b) the outputs and outcomes of this engagement. METHODS: We used a combination of one-on-one interviews with informal providers, group interviews with RIPEND project staff, and secondary analysis of data available within the project's recording and reporting systems. RESULTS: A total of 555 informal providers were actively engaged under the project. The majority (87%) had a nonmedicine-related graduate degree and had been providing medical care for more than 10 years. Most (95%) were aware that a cough for 2 weeks or more is a symptom of pulmonary TB and that such patients should be referred for sputum-smear microscopy at a government health facility. Challenges in engaging the informal providers included motivating them to participate in the study, suboptimal mobile usage for referral services, and delays in providing financial incentives to them for referring PPTBs. During the project period (October 2018-December 2019), 8342 PPTBs were identified of which 1003 TB patients were detected and linked to TB treatment services. CONCLUSION: This project showed that engaging informal providers is feasible and that a large number of PPTB and TB patients can be identified through this effort. The Government of India should consider engaging informal providers for the early diagnosis of TB to reduce the missing TB cases in the country.

Community health workers augment the cascade of TB detection to care in urban slums of two metro cities in India.

BACKGROUND: Tuberculosis Health Action Learning Initiative (THALI) funded by USAID is a person-centered initiative, supporting vulnerable urban populations to gain access to TB services. THALI trained and placed 112 Community health workers (CHWs) to detect and support individuals with TB symptoms or disease within urban slums in two cities, Hyderabad and Bengaluru, covering a population of about 3 million. METHODS: CHWs visited the slums once in a fortnight. They conducted TB awareness activities. They referred individuals with TB symptoms for sputum testing to nearest public sector laboratories. They visited those testing TB positive, once a fortnight in the intensive phase, and once a month thereafter. They supported TB patients and families with counselling, contact screening and social scheme linkages. They complemented the shortfall in urban TB government field staff numbers and their capacity to engage with TB patients. Data on CHWs' patient referral for TB diagnosis and treatment support activities was entered into a database and analyzed to examine CHWs' role in the cascade of TB care. We compared achievements of six monthly referral cohorts from September 2016 to February 2019. RESULTS: Overall, 31 617 (approximately 1%) of slum population were identified as TB symptomatic and referred for diagnosis. Among the referred persons, 23 976 (76%) underwent testing of which 3841 (16%) were TB positive. Overall, 3812 (99%) were initiated on treatment and 2760 (72%) agreed for regular follow up by the CHWs. Fifty-seven percent of 2952 referred were tested in the first cohort, against 86% of 8315 in the last cohort. The annualized case detection rate through CHW referrals in Bengaluru increased from 5.5 to 52.0 per 100 000 during the period, while in Hyderabad it was 35.4 initially and increased up to 118.9 per 100 000 persons. The treatment success rate was 87.1% among 193 in the first cohort vs 91.3% among 677 in the last cohort. CONCLUSIONS: CHWs in urban slums augment TB detection to care cascade. Their performance and TB treatment outcomes improve over time. It would be important to examine the cost per TB case detected and successfully treated.

Activity of natural and synthetic polygodial derivatives against Trypanosoma cruzi amastigotes, trypomastigotes and epimastigotes.

Our laboratories have been investigating biological effects of a sesquiterpenoid polygodial and its natural and synthetic analogues. Herein, we report the evaluation of these compounds against the three forms of Trypanosoma cruzi, amastigotes, trypomastigotes and epimastigotes. Although polygodial was found to be poorly active, its natural congener epipolygodial and synthetic Wittig-derived analogues showed low micromolar potency against all three forms of the parasite. Synthetic α,ß-unsaturated phosphonate 9 compared favorably with clinically approved drugs benznidazole and nifurtimox, and was effective against trypomastigotes, toward which benznidazole showed no activity.[Formula: see text].

Is a differentiated care model needed for patients with TB? A cohort analysis of risk factors contributing to unfavourable outcomes among TB patients in two states in South India.

BACKGROUND: TB is a preventable and treatable disease. Yet, successful treatment outcomes at desired levels are elusive in many national TB programs, including India. We aim to identify risk factors for unfavourable outcomes to TB treatment, in order to subsequently design a care model that would improve treatment outcomes among these at-risk patients. METHODS: We conducted a cohort analysis among TB patients who had been recently initiated on treatment. The study was part of the internal program evaluation of a USAID-THALI project, implemented in select towns/cities of Karnataka and Telangana, south India. Community Health Workers (CHWs) under the project, used a pre-designed tool to assess TB patients for potential risks of an unfavourable outcome. CHWs followed up this cohort of patients until treatment outcomes were declared. We extracted treatment outcomes from patient's follow-up data and from the Nikshay portal. The specific cohort of patients included in our study were those whose risk was assessed during July and September, 2018, subsequent to conceptualisation, tool finalisation and CHW training. We used bivariate and multivariate logistic regression to assess each of the individual and combined risks against unfavourable outcomes; death alone, or death, lost to follow up and treatment failure, combined as 'unfavourable outcome'. RESULTS: A significantly higher likelihood of death and experiencing unfavourable outcome was observed for individuals having more than one risk (AOR: 4.19; 95% CI: 2.47-7.11 for death; AOR 2.21; 95% CI: 1.56-3.12 for unfavourable outcome) or only one risk (AOR: 3.28; 95% CI: 2.11-5.10 for death; AOR 1.71; 95% CI: 1.29-2.26 for unfavourable outcome) as compared to TB patients with no identified risk. Male, a lower education status, an initial weight below the national median weight, co-existing HIV, previous history of treatment, drug-resistant TB, and regular alcohol use had significantly higher odds of death and unfavourable outcome, while age > 60 was only associated with higher odds of death. CONCLUSION: A rapid risk assessment at treatment initiation can identify factors that are associated with unfavourable outcomes. TB programs could intensify care and support to these patients, in order to optimise treatment outcomes among TB patients.

[Corrigendum] Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma.

After the publication of the above article, the authors have realized that they failed to properly acknowledge the work performed by their Center for Innovative Drug Discovery High Throughput Screening Core Facility in the paper. The Declarations section of their paper should therefore have also included the following statement: "The UT Health San Antonio Center for Innovative Drug Discovery HTS Facility was funded in part by the Cancer Prevention Research Institute of Texas (CPRIT; grant no. RP160844)". The authors regret their oversight in failing to include this information in the Declarations section of their paper. [the original article was published in International Journal of Oncology 53: 2627-2636, 2018; DOI: 10.3892/ijo.2018.4585].

Chemistry and biology of ophiobolin A and its congeners.

Ophiobolin A is a fungal secondary metabolite that was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, offering an innovative strategy to combat this aggressive cancer. The current article aims to make the bridge between the anti-cancer effects of ophiobolin A and its unique reaction with primary amines and suggests that pyrrolylation of lysine residues on its intracellular target protein(s) and/or phosphatidylethanolamine lipid is responsible for its biological effects. The article also discusses chemical derivatization of ophiobolin A to establish first synthetically generated structure-activity relationship. Finally, the reported total synthesis efforts toward the ophiobolin class of sesterterpenes are discussed and identified as a fertile area for improvement in pursuit of these molecules as anticancer agents.

Microtubule-Targeting 7-Deazahypoxanthines Derived from Marine Alkaloid Rigidins: Exploration of the N3 and N9 Positions and Interaction with Multidrug-Resistance Proteins.

Our laboratories have been investigating synthetic analogues of marine alkaloid rigidins that possess promising anticancer activities. These analogues, based on the 7-deazahypoxanthine skeleton, are available in one- or two-step synthetic sequences and exert cytotoxicity by disrupting microtubule dynamics in cancer cells. In the present work we extended the available structure-activity relationship (SAR) data to N3- and N9-substituted derivatives. Although N3 substitution results in loss of activity, the N9-substituted compounds retain nanomolar antiproliferative activities and the anti-tubulin mode of action of the original unsubstituted compounds. Furthermore, our results also demonstrate that multidrug-resistance (MDR) proteins do not confer resistance to both N9-unsubstituted and -substituted compounds. It was found that sublines overexpressing ABCG2, ABCC1, and ABCB1 proteins are as responsive to the rigidin analogues as their parental cell lines. Thus, the study reported herein provides further impetus to investigate the rigidin-inspired 7-deazahypoxanthines as promising anticancer agents.

Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma.

Polygodial, a drimane sesquiterpenoid dialdehyde isolated as a pungent component of the water pepper Persicaria hydropiper, exhibits antifeedant, antimicrobial, anti-inflammatory and anticancer effects. Polygodial also activates transient receptor potential vanilloid subtype 1 (TRPV1) channels. Previously, we described the synthesis of a C12-Wittig derivative of polygodial, termed P3, with significant antiproliferative effects against multiple cancer types including oral squamous cell carcinoma (OSCC). In the present study, a more potent derivative, P27, with superior anti-proliferative effects in vitro and antitumor effects in Cal-27 derived xenografts is described. Polygodial, P3, and P27 all significantly decreased OSCC tumor growth, with P27 being equipotent with polygodial and P3 being the least efficacious. However, neither analog elicited the adverse effect observed with polygodial: Profound transient inflammation. Although P3 and P27 pharmacophores are based on polygodial, novel effects on OSCC cell cycle distribution were identified and shared anticancer effects that are independent of TRPV1 activity were observed. Polygodial elicits an S-phase block, whereas P3 and P27 lead to G2/M phase arrest. Pretreatment of OSCC cells with the TRPV1 antagonist capsazepine does not affect the antiproliferative activity of P3 or P27, indicating that TRPV1 interactions do not regulate OSCC cell proliferation. Indeed, calcium imaging studies identified that the analogs neither activate nor antagonize TRPV1. Behavioral studies using a rat model for orofacial pain confirmed that these analogs fail to induce nocifensive responses, indicating that they are non-noxious in vivo. All compounds induced a significant concentration-dependent decrease in the mitochondrial transmembrane potential and corresponding apoptosis. Considering that P27 is equipotent to polygodial with no TRPV1-associated adverse effects, P27 may serve as an efficacious novel therapy for OSCC.

Effect of polygodial and its direct derivatives on the mammalian Na+/K+-ATPase activity.

The sesquiterpene polygodial is an agonist of the transient receptor potential vanilloid 1 (TRPV1). Our group recently reported the synthesis and anticancer effects of polygodial and its derivatives, and showed that these compounds retain activity against apoptosis- and multidrug-resistant cancer cells. Herein, we tested the inhibitory effect of these compounds on the activity of the enzyme Na+/K+-ATPase (NKA) from kidney (α1 isoform) and brain (α2 and α3 isoforms) guinea pig extracts. Polygodial (1) displayed a dose-dependent inhibition of both kidney and brain purified NKA preparations, with higher sensitivity for the cerebral isoforms. Polygo-11,12-diol (2) and C11,C12-pyridazine derivative (3) proved to be poor inhibitors. Unsaturated ester (4) and 9-epipolygodial (5) inhibited NKA preparations from brain and kidney, with the same inhibitory potency. Nevertheless, they did not achieve maximum inhibition even at higher concentration. Comparing the inhibitory potency in crude homogenates and purified preparations of NKA, compounds 4 and 5 revealed a degree of selectivity toward the renal enzyme. Kinetic studies showed a non-competitive inhibition for Na+ and K+ by compounds 1, 4 and 5 and for ATP by 1 and 4. However, compound 5 presented a competitive inhibition type. Furthermore, K+-activated p-nitrophenylphosphatase activity of these purified preparations was not inhibited by 1, 4 and 5, suggesting that these compounds acted in the initial phase of the enzyme's catalytic cycle. These findings suggest that the antitumor action of polygodial and its analogues may be linked to their NKA inhibitory properties and reinforce that NKA may be an important target for cancer therapy.

Polygodial analog induces apoptosis in LNCaP prostate cancer cells.

Prostate cancer (PCa) is the second leading cause of death in American men. The chemotherapeutic treatment strategies are generally not effective and can lead to side effects. Hence, there is an urgent need to identify novel chemotherapeutic agents. The aim of this study was to synthesize and evaluate the therapeutic effects of a synthetic analog of polygodial (PG), a pungent constituent abundantly present in mountain pepper, water pepper and dorrigo pepper, on LNCaP PCa cell line and its anti-cancer mechanisms in a preclinical study. We evaluated the anti-cancer potential of the PG analog namely DRP-27 using various assays such as cell viability by MTT assay, anchorage independent growth by soft agar assay, reactive oxygen species generation by 2',7'-dichlorofluorescein probe-based fluorescence assay, and apoptosis by Annexin-V and TUNEL assays respectively. Western blot analysis was performed to identify the molecular mechanism of DRP-27-induced cell death. Our results showed that DRP-27 significantly inhibited LNCaP cell proliferation in a dose-dependent manner at 48 h treatment in vitro. In addition, DRP-27 potently inhibited anchorage-independent growth of these cells. Flow cytometry, Annexin-V and TUNEL assays confirmed that DRP-27 induces apoptosis in LNCaP cells. DRP-27 also induced the activation of intracellular reactive oxygen species. Western blot analysis revealed that DRP-27 downregulated the expression of survivin, while activating Bax and DNA damage marker pH2AX in LNCaP cells. In conclusion, our study suggests that DRP-27 might be an effective anti-cancer agent for PCa.

Irreversible Protein Labeling by Paal-Knorr Conjugation.

The application of new chemical reactions in a biological context has advanced bioconjugation methods for both fundamental research and commercial arenas. Recent adaptations of reactions such as Huisgen 1,3-dipolar or Diels-Alder cycloadditions have enabled the labeling of specific residues in biomolecules by the attachment of molecules carrying azides, alkynes, or strained alkenes. Although these are fundamental tools, there is a need for the discovery of reactions that can label native proteins. We report herein the adaptation of the Paal-Knorr reaction to label lysine residues in proteins via pyrrole linkages.

Marine Mollusk-Derived Agents with Antiproliferative Activity as Promising Anticancer Agents to Overcome Chemotherapy Resistance.

The chemical investigation of marine mollusks has led to the isolation of a wide variety of bioactive metabolites, which evolved in marine organisms as favorable adaptations to survive in different environments. Most of them are derived from food sources, but they can be also biosynthesized de novo by the mollusks themselves, or produced by symbionts. Consequently, the isolated compounds cannot be strictly considered as "chemotaxonomic markers" for the different molluscan species. However, the chemical investigation of this phylum has provided many compounds of interest as potential anticancer drugs that assume particular importance in the light of the growing literature on cancer biology and chemotherapy. The current review highlights the diversity of chemical structures, mechanisms of action, and, most importantly, the potential of mollusk-derived metabolites as anticancer agents, including those biosynthesized by mollusks and those of dietary origin. After the discussion of dolastatins and kahalalides, compounds previously studied in clinical trials, the review covers potentially promising anticancer agents, which are grouped based on their structural type and include terpenes, steroids, peptides, polyketides and nitrogen-containing compounds. The "promise" of a mollusk-derived natural product as an anticancer agent is evaluated on the basis of its ability to target biological characteristics of cancer cells responsible for poor treatment outcomes. These characteristics include high antiproliferative potency against cancer cells in vitro, preferential inhibition of the proliferation of cancer cells over normal ones, mechanism of action via nonapoptotic signaling pathways, circumvention of multidrug resistance phenotype, and high activity in vivo, among others. The review also includes sections on the targeted delivery of mollusk-derived anticancer agents and solutions to their procurement in quantity.

Single dish gradient screening of small molecule localization.

Understanding trafficking in cells and tissues is one of the most critical steps in exploring the mechanisms and modes of action (MOAs) of a small molecule. Typically, deciphering the role of concentration presents one of the most difficult challenges associated with this task. Herein, we present a practical solution to this problem by developing concentration gradients within single dishes of cells. We demonstrate the method by evaluating fluorescently-labelled probes developed from two classes of natural products that have been identified as potential anti-cancer leads by STORM super-resolution microscopy.

5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells.

Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-type alkaloids based on the 5,10b-ethanophenanthridine ring system were recently shown to be effective at inhibiting proliferation of cancer cells resistant to various pro-apoptotic stimuli and representing tumors with dismal prognoses refractory to current chemotherapy, such as glioma, melanoma, non-small-cell lung, esophageal, head and neck cancers, among others. Using this discovery as a starting point and taking advantage of a concise biomimetic route to the crinine skeleton, a collection of crinine analogues were synthetically prepared and evaluated against cancer cells. The compounds exhibited single-digit micromolar activities and retained this activity in a variety of drug-resistant cancer cell cultures. This investigation resulted in the discovery of new bicyclic ring systems with significant potential in the development of effective clinical cancer drugs capable of overcoming cancer chemotherapy resistance.

Marine Invertebrate Metabolites with Anticancer Activities: Solutions to the "Supply Problem".

Marine invertebrates provide a rich source of metabolites with anticancer activities and several marine-derived agents have been approved for the treatment of cancer. However, the limited supply of promising anticancer metabolites from their natural sources is a major hurdle to their preclinical and clinical development. Thus, the lack of a sustainable large-scale supply has been an important challenge facing chemists and biologists involved in marine-based drug discovery. In the current review we describe the main strategies aimed to overcome the supply problem. These include: marine invertebrate aquaculture, invertebrate and symbiont cell culture, culture-independent strategies, total chemical synthesis, semi-synthesis, and a number of hybrid strategies. We provide examples illustrating the application of these strategies for the supply of marine invertebrate-derived anticancer agents. Finally, we encourage the scientific community to develop scalable methods to obtain selected metabolites, which in the authors' opinion should be pursued due to their most promising anticancer activities.

Novel Microtubule-Targeting 7-Deazahypoxanthines Derived from Marine Alkaloid Rigidins with Potent in Vitro and in Vivo Anticancer Activities.

Docking studies of tubulin-targeting C2-substituted 7-deazahypoxanthine analogues of marine alkaloid rigidins led to the design and synthesis of compounds containing linear C2-substituents. The C2-alkynyl analogue was found to have double- to single-digit nanomolar antiproliferative IC50 values and showed statistically significant tumor size reduction in a colon cancer mouse model at nontoxic concentrations. These results provide impetus and further guidance for the development of these rigidin analogues as anticancer agents.

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9-Epipolygodial against Drug-Resistant Cancer Cells.

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1-agonist and anticancer activities. These experiments led to the identification of 9-epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9-Epipolygodial was found to maintain potency against apoptosis-resistant cancer cells as well as those displaying the multidrug-resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal-Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Fungal metabolite ophiobolin A as a promising anti-glioma agent: In vivo evaluation, structure-activity relationship and unique pyrrolylation of primary amines.

Glioblastoma, the most common form of malignant primary brain tumor, is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches based on apoptosis induction in cancer cells. Previously, a fungal secondary metabolite ophiobolin A was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, thus, offering an innovative strategy to combat this type of cancer. The current work describes the results of a preliminary evaluation of ophiobolin A in an in vivo glioblastoma model and its chemical derivatization to establish first synthetically generated structure-activity relationship. The synthetic work has also led to the discovery of a unique reaction of ophiobolin A with primary amines suggesting the possibility of pyrrolylation of lysine residues on its intracellular target protein(s).