Cellular and Molecular Mechanisms Underlying Scope and Limitation of Ongoing and Innovative Therapies for Treating Chronic Hepatitis B

Millions of people of the world suffer from chronic hepatitis B (CHB), a pathological entity in which the patients are chronically infected with hepatitis B virus (HBV) and express hepatitis B surface antigen (HBsAg) and HBV DNA, as well as evidence of liver damages. Considerable numbers of CHB patients develop cirrhosis of the liver and hepatocellular carcinoma if untreated. Two groups of drugs (interferons and nucleoside analogs) are used to treat CHB patients, but both are endowed with considerable adverse effects, increased costs, extended duration of therapy, and limited efficacy. Thus, there is a pressing need to develop new and innovative therapeutics for CHB patients, and many such drugs have been developed during the last four decades. Some of these drugs have inspired considerable optimism to be a game-changer for the treatment of CHB. Here, we first discuss why ongoing therapeutics such as interferon and nucleoside analogs could not stand the test of time. Next, we dissect the scope and limitation of evolving therapies for CHB by dissecting the cellular and molecular mechanisms of some of these innovative therapeutics.

Recapitulating Trafficking of Nucleosides Into the Active Site of Polymerases of RNA Viruses: The Challenge and the Prize

Nucleoside analogs are very effective antiviral agents with currently over 25 compounds approved for the therapy of viral infections. Still, their successful use against RNA viruses is very recent, despite RNA viruses comprising some of the most damaging human pathogens (e.g., Coronaviruses, Influenza viruses, or Flaviviridae such as dengue, Zika and hepatitis C viruses). The breakthrough came in 2013–2014, when the nucleoside analog Sofosbuvir became one of the cornerstones of current curative treatments for hepatitis C virus (HCV). An analog designed on the same principles, Remdesivir, has been the first approved compound against SARS-CoV-2, the coronavirus that causes the current COVID-19 pandemic. Both of these nucleoside analogs target the RNA-dependent RNA polymerase (RdRp) (NS5B for HCV, nsp12 for SARS-CoV-2). RdRps of RNA viruses display a peculiar elaboration of the classical polymerase architecture that leads to their active site being caged. Thus, triphosphate nucleosides and their analogs must access this active site in several steps along a narrow and dynamic tunnel. This makes straightforward computational approaches such as docking unsuitable for getting atomic-level details of this process. Here we give an account of ribose-modified nucleoside analogs as inhibitors of viral RdRps and of why taking into account the dynamics of these polymerases is necessary to understand nucleotide selection by RdRps. As a case study we use a computational protocol we recently described to examine the approach of the NTP tunnel of HCV NS5B by cellular metabolites of Sofosbuvir. We find major differences with natural nucleotides even at this early stage of nucleotide entry.

Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH)

Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme’s ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

Impact of SAMHD1 Pharmacogenetics on Clinical Outcome in Pediatric AML

Acute myeloid leukemia (AML) is a heterogeneous disease characterized in part by genetic and epigenetic alterations. Cytarabine arabinoside (Ara-C) has been the cornerstone of chemotherapy treatment for patients diagnosed with AML for decades. Following cellular uptake, ara-C is phosphorylated into its active metabolite, ara-CTP, which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in ara-C activation/inactivation pathway (Fig 1A), can impact intracellular abundance of ara-CTP and thus its therapeutic benefit. Recently, deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) was shown to limit the efficacy of ara-C by intracellularly hydrolyzing its active metabolite (PMID: 27991919). Other nucleoside analogs, such as clofarabine, fludarabine, and gemcitabine have also been shown to be substrates of SAMHD1 (PMID:30305425). Among adult AML patients, higher SAMHD1 expression in the leukemic cells has been found to correlate with poor outcome (PMID: 30341277). However, whether genetic variation in SAMHD1 has any impact on the clinical outcomes in pediatric AML patients has not been evaluated in depth. In this study, we looked into 25 single nucleotide polymorphisms (SNPs) within SAMHD1 gene for their association with clinical outcome of newly diagnosed pediatric AML patients in 2 cohorts (multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=231] clinical trials). Briefly, genotypes for the 25 SNPs within SAMHD1 genes were obtained from Illumina 2.5 Omni data of AML patients. Association analysis was conducted using logistic regression models comparing minimal residual disease (MRD) positivity after treatment with chemotherapy using an unadjusted model and an adjusted model stratified by initial risk assignment of the patient determined at diagnosis (i.e. low, standard, and high). MRD positivity was defined as one or more leukemic cell per 1000 mononuclear bone-marrow cells. Cox proportional hazard models were used to examine the association of the SNPs with EFS and OS and considered three modes of inheritance. Cox regression models were performed with or without adjusting for provisional risk group assignment at time of diagnosis. Significance levels for association of SNP with clinical outcome were set at p < 0.05. Three top SNPs significantly associated with clinical outcomes were all localized in the 3'UTR region of SAMHD1(Fig 1B). Presence of the variant allele of rs7265241 was associated with a lower EFS and OS in both AML02 and AML08 cohorts. Figure 1C shows the results for OS in AML02 (HR = 2.24, 95% CI (1.05-3.55), p=0.02) and AML08 (HR = 1.52, 95% CI (1.04-1.99), p=0.01). Another 3'UTR SNP, rs1291128 was associated with poor OS in AML02 and in the clofarabine plus ara-C treatment arm in AML08 trial. For rs1291141, T allele was associated with higher MRD positivity (OR: 2.12, p=0.01) in AML02 and poor OS (HR = 1.55, 95% CI (1.12-2.14), p=0.008) in AML08(Fig 1D). Consistent with the clinical outcome results, rs1291141 T allele was also associated with higher SAMHD1 expression in the whole blood (p=8.1e-14) and several other tissues in the GTEx database. https://gtexportal.org/home/ Our results suggest that genetic variation in SAMHD1 is associated with clinical outcomes in pediatric AML patients. Future studies are aimed at looking at haplotypes and SNP-SNP combinations to establish the impact SAMHD1 pharmacogenomics on its expression within leukemic cells and subsequent response to nucleoside analogs in AML patients. SAMHD1 has also been implicated as a tumor suppressor; thus, future studies will evaluate its role both as a proliferation regulator and in drug resistance. Acknowledgements: NIH-R01-CA132946 (Lamba and Pounds), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC), and American Society of Hematology Bridge funding (Lamba) funded the study. We thank Drs. Campana, Coustan-Smith, and Raimondi for MRD and cytogenetic data. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Purine Nucleoside Analogs Plus Rituximab Is an Effective Treatment Choice for Hairy Cell Leukemia-Variant: Results from a Single Center in China

Background: Hairy cell leukemia-variant (HCL-v) is one type of chronic lymphocytic proliferative disorders which was classified into splenic B-cell lymphoma/leukemia, unclassifiable. Both clinical and laboratory characteristics and treatment strategy remain elusive due to the rarity of the disease. Here, we firstly presented the diseases features and efficacy of a variety of treatment options in a large cohort from China. Methods: Thirty-three patients were diagnosed with HCL-v in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College from June 1990 to February 2021. We analyzed the disease characteristics and treatment outcome, especially in terms of clinical manifestation, immunophenotypic and molecular evaluation and efficacy of multiple first-line treatment. Results: The median age of this cohort was 59 years (range, 34-79 years) at diagnosis, with 23 males and 10 females. Abdominal mass and relative signs (n=22) and abnormal complete blood count (n=9) were the most common chief complaints. Splenomegaly was present in 32 (97.0%) cases, among whom 26 (78.8%) cases were massive. Leukocytosis and leukopenia were presented in 23 (69.7%) patients and 5 (15.2%) patients, with a median white blood cell count of 21.58×10 9/L (range, 1.34-224.59×10 9/L). The median percentage of the leukemic cells in bone marrow tested by flow cytometry was 33% (range, 6.2%-96%), and immunophenotyping showed monoclonal B-cells positive for pan B-cell antigens (CD19, CD20, CD22, CD79b) in almost all patients (Table). CD11c was positive in all patients, and CD103 was positive in 20/26 (76.9%) patients. CD25 was negative in 30/33 (90.9%) patients, and CD23 was negative in 20/26 (76.9%) cases. CD200 was frequently expressed among patients (15/21, 71.4%). For the pathological pattern of bone marrow involvement, 20/27 (74.1%) patients showed a predominantly interstitial pattern. Moreover, Annexin A1 was negative in all (n=12) patients detected by immunohistochemistry. Conventional cytogenetic analysis showed abnormal karyotype in 10/24 (41.7%) patients, including 6 patients with complex karyotype. Fluorescence in situ hybridization analysis showed deletion of TP53 in 4/20 (20.0%) patients and IGH translocation in 5/16 (31.3%) patients. The BRAF V600E mutation was negative in all the patients (n=20). In 2 of 14 (16.7%) patients, TP53 mutation was detected by next-generation sequencing. Sixteen of nineteen (84.2%) patients showed monoclonal IGHV rearrangements, and the most common rearrangement fragment was VH4-34 (n=3, 18.8%). Six of seventeen (35.3%) patients presented with unmutated IGHV and patients with VH4-34 were all unmutated. 31 patients needed treatment finally. Treatment choices included interferon-α (IFN-α) in 11 patients, chlorambucil (CLB) in 5 patients, single purine nucleoside analogs (PNA) in 3 patients and PNA plus rituximab in 9 patients. Four patients who received IFN-α or CLB treatment also underwent splenectomy. The objective response rate was 100% for patients receiving PNA plus rituximab and 77% for cases receiving others. And people treated with PNA plus rituximab had a higher complete response rate compared to the other regimen (75% versus 12%, P = 0.004). During a median follow-up of 32 months (range, 3-207) for 29 patients, 14 (48.3%) patients experienced at least one relapse or progression, and 7 (24.1%) patients had 2 or more relapses. Of note, one case also underwent a diffuse large B-cell lymphoma transformation. The median PFS and OS were 31 months (95% CI 25.5-36.5) and 70 months (95% CI 50.5-89.5), respectively. PNA plus rituximab prolong PFS compared to the others (3-year PFS rate 80% [95%CI 20-97] versus 10% [95%CI 1-35], P = 0.012, Figure A). But no significant difference in 3-year OS rate was observed between two groups (100% [95%CI 100-100] versus 43% [95%CI 10-73], P = 0.128, Figure B). Conclusion: HCL-v is a rare disease with specific clinical and immunophenotypic features but may overlap with classic hairy cell leukemia or other splenic B-cell neoplasms. Overall, it is an indolent lymphoma with recurrent progression, and the use of PNA plus rituximab in first-line treatment can result in a deeper and longer remission. Figure 1 Figure 1. Disclosures Wang: AbbVie: Consultancy; Astellas Pharma, Inc.: Research Funding.

Variability in the response of HBV D-subgenotypes to antiviral therapy: designing pan D-subgenotypic reverse transcriptase inhibitors

Nucleos(t)ide analogues entecavir (ETV ) and tenofovir disoproxil fumarate (TDF ) are recommended as first - line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3 - fluoro - 2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N 6 -substituted ( S )-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized ( S )-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. Importance: Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response towards TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

Generative AI Design and Exploration of Nucleoside Analogs

Nucleosides are fundamental building blocks of DNA and RNA in all life forms and viruses. In addition, natural nucleosides and their analogs are critical in prebiotic chemistry, innate immunity, signaling, antiviral drug discovery and artificial synthesis of DNA / RNA sequences. Combined with the fact that quantitative structure activity relationships (QSAR) have been widely performed to understand their antiviral activity, nucleoside analogs could be used to benchmark generative molecular design. Here, we undertake the first generative design of nucleoside analogs using an approach that we refer to as the Conditional Randomized Transformer (CRT). We also benchmark our model against five previously published molecular generative models. We demonstrate that AI-generated molecules include nucleoside analogs that are of significance in a wide range of areas including prebiotic chemistry, antiviral drug discovery and synthesis of oligonucleotides. Our results show that CRT explores distinct molecular spaces and chemical transformations, some of which are similar to those undertaken by nature and medicinal chemists. Finally, we demonstrate the potential application of the CRT model in the generative design of molecules conditioned on Remdesivir and Molnupiravir as well as other nucleoside analogs with in vitro activity against SARS-CoV-2. One-Sentence Summary: Generative design of nucleoside analogs relevant to antiviral drug discovery, prebiotic chemistry and synthetic biology.

1404. Tuberculosis and HIV Coinfection: A Review of 135 Cases Experience of the Infectious Diseases Department- CHU Mohamed VI- Marrakech

Background Tuberculosis is a health problem in Morocco, which is increasingly indicative of human immunodeficiency virus (HIV) infection. Objective To determine the epidemiological, clinical and paraclinical, therapeutic and evolutionary aspects of tuberculosis and HIV co-infection. Methods we report 135 cases co-infected with HIV and tuberculosis, collected by the infectious diseases department at the Mohammed VI University Hospital in Marrakech. This is a 12-year retrospective study (2007 to 2020) that involved all HIV-infected patients hospitalized for tuberculosis regardless of its location. Results The mean age of the patients was 40 years (17-73 years). A male predominance was noted in 69% of cases. In 74.6% of cases, tuberculosis was indicative of HIV infection. Nine patients were receiving antiretroviral (ARV) treatment at the time of the discovery of tuberculosis. There were 24% pulmonary tuberculosis, 25.3% extrapulmonary tuberculosis and 49% disseminated tuberculosis. Tuberculosis was confirmed in 31.7% of cases. At the time of tuberculosis diagnosis, the average CD4 count was 86 cells / mm. Quadruple therapy with isoniazid, rifampicin, pyrazinamide and ethambutol was started in 83% of patients. The average time to start ARVs was 7 weeks. All patients who received ARVs received a combination therapy comprising the combination of 2 nucleoside analogs and one non-nucleoside analog. At the end of our work, the evolution was favorable in 53% of cases, death occurred in 25% of cases, 18.6% of patients were lost to follow-up, two cases of failure and another of relapse. Immune restoration syndrome was noted in 8 cases. Drug toxicity was observed in 24.5% of patients, 73% of which was related to hepato-toxicity of antibacillary drugs. Conclusion Tuberculosis is the most common opportunistic infection in people with HIV. Despite the advent of highly active triple therapy, tuberculosis is still a major cause of death in HIV positive people. Disclosures All Authors: No reported disclosures

A Combination of Amino Acid Mutations Leads to Resistance to Multiple Nucleoside Analogs in Reverse Transcriptases from HIV-1 Subtypes B and C

Resistance to anti-Human Immunodeficiency Virus (HIV) drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had a low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT or Zidovudine), and (−)-L-2′,3′-dideoxy-3′-thiacytidine (Lamivudine or 3TC). The RT had mutations at positions 64, 67, 70, 184, 219, and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside reverse transcriptase inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by Tenofovir.