Antigen-Clustered Nanovaccine Achieves Long-Term Tumor Remission by Promoting B/CD 4 T Cell Crosstalk.

Current cancer vaccines using T cell epitopes activate antitumor T cell immunity through dendritic cell/macrophage-mediated antigen presentation, but they lack the ability to promote B/CD4 T cell crosstalk, limiting their anticancer efficacy. We developed antigen-clustered nanovaccine (ACNVax) to achieve long-term tumor remission by promoting B/CD4 T cell crosstalk. The topographic features of ACNVax were achieved using an iron nanoparticle core attached with an optimal number of gold nanoparticles, where the clusters of HER2 B/CD4 T cell epitopes were conjugated on the gold surface with an optimal intercluster distance of 5-10 nm. ACNVax effectively trafficked to lymph nodes and cross-linked with BCR, which are essential for stimulating B cell antigen presentation-mediated B/CD4 T cell crosstalk in vitro and in vivo. ACNVax, combined with anti-PD-1, achieved long-term tumor remission (>200 days) with 80% complete response in mice with HER2+ breast cancer. ACNVax not only remodeled the tumor immune microenvironment but also induced a long-term immune memory, as evidenced by complete rejection of tumor rechallenge and a high level of antigen-specific memory B, CD4, and CD8 cells in mice (>200 days). This study provides a cancer vaccine design strategy, using B/CD4 T cell epitopes in an antigen clustered topography, to achieve long-term durable anticancer efficacy through promoting B/CD4 T cell crosstalk.

Discovery of CBPD-268 as an Exceptionally Potent and Orally Efficacious CBP/p300 PROTAC Degrader Capable of Achieving Tumor Regression.

CBP/p300 proteins are key epigenetic regulators and promising targets for the treatment of castration-resistant prostate cancer and other types of human cancers. Herein, we report the discovery and characterization of CBPD-268 as an exceptionally potent, effective, and orally efficacious PROTAC degrader of CBP/p300 proteins. CBPD-268 induces CBP/p300 degradation in three androgen receptor-positive prostate cancer cell lines, with DC50 ≤ 0.03 nM and Dmax > 95%, leading to potent cell growth inhibition. It has an excellent oral bioavailability in mice and rats. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent CBP/p300 depletion in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taking these results together, CBPD-268 is a highly promising CBP/p300 degrader as a potential new cancer therapy.

Discovery of CBPD-409 as a Highly Potent, Selective, and Orally Efficacious CBP/p300 PROTAC Degrader for the Treatment of Advanced Prostate Cancer.

CBP/p300 are critical transcriptional coactivators of the androgen receptor (AR) and are promising cancer therapeutic targets. Herein, we report the discovery of highly potent, selective, and orally bioavailable CBP/p300 degraders using the PROTAC technology with CBPD-409 being the most promising compound. CBPD-409 induces robust CBP/p300 degradation with DC50 0.2-0.4 nM and displays strong antiproliferative effects with IC50 1.2-2.0 nM in the VCaP, LNCaP, and 22Rv1 AR+ prostate cancer cell lines. It has a favorable pharmacokinetic profile and achieves 50% of oral bioavailability in mice. A single oral administration of CBPD-409 at 1 mg/kg achieves >95% depletion of CBP/p300 proteins in the VCaP tumor tissue. CBPD-409 exhibits strong tumor growth inhibition and is much more potent and efficacious than two CBP/p300 inhibitors CCS1477 and GNE-049 and the AR antagonist Enzalutamide. CBPD-409 is a promising CBP/p300 degrader for further extensive evaluations for the treatment of advanced prostate cancer and other types of human cancers.

Bioanalysis of six antibiotics from volumetric microsamples: a new tool for precision dosing in critically ill children.

Background: Volumetric absorptive microsamples (VAMS) can support pharmacokinetic / pharmacodynamic studies. We present the bioanalytical method development for the simultaneous quantification of ampicillin, cefepime, ceftriaxone, meropenem, piperacillin, tazobactam, and vancomycin from VAMS. Methods & results: Optimal extraction, chromatographic, and mass spectrometry conditions were identified. Maximum extraction recoveries included 100 µl of water for rehydration and methanol for protein precipitation. Chromatographic separation used Phenomenex Kinetex™ Polar C18 column with a mobile phase comprising water/acetonitrile with formic acid and was fully validated. Hematocrit effects were only observed for vancomycin. Samples were stable for 90 days at -80°C except for meropenem, which was stable for 60 days. Conclusion: Multiple antibiotics can be assayed from a single VAMS sample to facilitate pharmacokinetic/pharmacodynamic studies.

A gastrointestinal locally activating Janus kinase inhibitor to treat ulcerative colitis.

In this study, we integrated machine learning (ML), structure-tissue selectivity-activity-relationship (STAR), and wet lab synthesis/testing to design a gastrointestinal (GI) locally activating JAK inhibitor for ulcerative colitis treatment. The JAK inhibitor achieves site-specific efficacy through high local GI tissue selectivity while minimizing the requirement for JAK isoform specificity to reduce systemic toxicity. We used the ML model (CoGT) to classify whether the designed compounds were inhibitors or noninhibitors. Then we used the regression ML model (MTATFP) to predict their IC50 against related JAK isoforms of predicted JAK inhibitors. The ML model predicted MMT3-72, which was retained in the GI tract, to be a weak JAK1 inhibitor, while MMT3-72-M2, which accumulated in only GI tissues, was predicted to be an inhibitor of JAK1/2 and TYK2. ML docking methods were applied to simulate their docking poses in JAK isoforms. Application of these ML models enabled us to limit our synthetic efforts to MMT3-72 and MMT3-72-M2 for subsequent wet lab testing. The kinase assay confirmed MMT3-72 weakly inhibited JAK1, and MMT3-72-M2 inhibited JAK1/2 and TYK2. We found that MMT3-72 accumulated in the GI lumen, but not in GI tissue or plasma, but released MMT3-72-M2 accumulated in colon tissue with minimal exposure in the plasma. MMT3-72 achieved superior efficacy and reduced p-STAT3 in DSS-induced colitis. Overall, the integration of ML, the structure-tissue selectivity-activity-relationship system, and wet lab synthesis/testing could minimize the effort in the optimization of a JAK inhibitor to treat colitis. This site-specific inhibitor reduces systemic toxicity by minimizing the need for JAK isoform specificity.

Prophylactic Mitigation of Acute Graft versus Host Disease by Novel 2-(Pyrrolidin-1-ylmethyl)pyrrole-Based Stimulation-2 (ST2) Inhibitors.

Hematopoietic cell transplantation (HCT) is a proven and potentially curable therapy for hematological malignancies and inherited hematological disease. The main risk of HCT is the development of graft versus host disease (GVHD) acquired in up to 50% of patients. Upregulation of soluble ST2 (sST2) is a key clinical biomarker for GVHD prognosis and was shown to be a potential therapeutic target for GVHD. Agents targeting sST2 to reduce the sST2 level after HCT have the potential to mitigate GVHD progression. Here, we report 32 (or XY52) as the lead ST2 inhibitor from our optimization campaign. XY52 had improved inhibitory activity and metabolic stability in vitro and in vivo. XY52 suppressed proinflammatory T-cell proliferation while increasing regulatory T cells in vitro. In a clinically relevant GVHD model, a 21-day prophylactic regimen of XY52 reduced plasma sST2 and IFN-γ levels and GVHD score and extended survival in mice. XY52 represented a significant improvement over our previous compound, iST2-1, and further optimization of XY52 is warranted. The small-molecule ST2 inhibitors can potentially be used as a biomarker-guided therapy for mitigating GVHD in future clinical applications.

Discovery of ARD-1676 as a Highly Potent and Orally Efficacious AR PROTAC Degrader with a Broad Activity against AR Mutants for the Treatment of AR + Human Prostate Cancer.

We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.

Discovery of ERD-3111 as a Potent and Orally Efficacious Estrogen Receptor PROTAC Degrader with Strong Antitumor Activity.

Estrogen receptor α (ERα) is a prime target for the treatment of ER-positive (ER+) breast cancer. Despite the development of several effective therapies targeting ERα signaling, clinical resistance remains a major challenge. In this study, we report the discovery of a new class of potent and orally bioavailable ERα degraders using the PROTAC technology, with ERD-3111 being the most promising compound. ERD-3111 exhibits potent in vitro degradation activity against ERα and demonstrates high oral bioavailability in mice, rats, and dogs. Oral administration of ERD-3111 effectively reduces the levels of wild-type and mutated ERα proteins in tumor tissues. ERD-3111 achieves tumor regression or complete tumor growth inhibition in the parental MCF-7 xenograft model with wild-type ER and two clinically relevant ESR1 mutated models in mice. ERD-3111 is a promising ERα degrader for further extensive evaluations for the treatment of ER+ breast cancer.

Discovery of SMD-3040 as a Potent and Selective SMARCA2 PROTAC Degrader with Strong in vivo Antitumor Activity.

SMARCA2 is an attractive synthetic lethality target for human cancers with SMARCA4 deficiency. Herein, we report the design, synthesis, and biological evaluation of selective SMARCA2 protein degraders developed using the proteolysis targeting chimera (PROTAC) technology. Our efforts have led to the discovery of a series of potent and selective SMARCA2 degraders, exemplified by SMD-3040. SMD-3040 degrades SMARCA2 protein with a low nanomolar DC50 and Dmax > 90% and demonstrates an excellent degradation selectivity for SMARCA2 protein over SMARCA4 protein. It displays potent cell growth inhibitory activity in a panel of SMARCA4-deficient cancer cell lines and has much weaker activity in SMARCA4 wild-type cancer cell lines. SMD-3040 achieves strong tumor growth inhibition in two SMARCA4-deficient xenograft models at well-tolerated dose schedules. Further optimization of SMD-3040 may lead to the discovery of new therapies for the treatment of human cancers with SMARCA4 deficiency.

Stochastic Differential Equation-based Mixed Effects Model of the Fluid Volume in the Fasted Stomach in Healthy Adult Human.

The rate and extent of drug dissolution and absorption from a solid oral dosage form depend largely on the fluid volume along the gastrointestinal tract. Hence, a model built upon the gastric fluid volume profiles can help to predict drug dissolution and subsequent absorption. To capture the great inter- and intra-individual variability (IAV) of the gastric fluid volume in fasted human, a stochastic differential equation (SDE)-based mixed effects model was developed and compared with the ordinary differential equation (ODE)-based model. Twelve fasted healthy adult subjects were enrolled and had their gastric fluid volume measured before and after consumption of 240 mL of water at pre-determined intervals for up to 2 hours post ingestion. The SDE- and ODE-based mixed effects models were implemented and compared using extended Kalman filter algorithm via NONMEM. The SDE approach greatly improved the goodness of fit compared with the ODE counterpart. The proportional and additive measurement error of the final SDE model decreased from 14.4 to 4.10% and from 17.6 to 4.74 mL, respectively. The SDE-based mixed effects model successfully characterized the gastric volume profiles in the fasted healthy subjects, and provided a robust approximation of the physiological parameters in the very dynamic system. The remarkable IAV could be further separated into system dynamics terms and measurement error terms in the SDE model instead of only empirically attributing IAV to measurement errors in the traditional ODE method. The system dynamics were best captured by the random fluctuations of gastric emptying coefficient Kge.

Discovery of Exceptionally Potent, Selective, and Efficacious PROTAC Degraders of CBP and p300 Proteins.

The histone acetyltransferase CREB-binding protein (CBP) and its paralogue p300 protein are key transcriptional coactivators and attractive cancer therapeutic targets. We describe herein our design, synthesis, and extensive evaluation of exceptionally potent PROTAC degraders of CBP/p300, exemplified by JET-209 (24). This compound, JET-209, achieved a half-maximal degradation (DC50) value of 0.05 nM for CBP and 0.2 nM for p300 with maximum degradation (Dmax) >95% for both proteins in the RS4;11 leukemia cell line after 4 h of treatment. JET-209 achieved subnanomolar to low nanomolar DC50 values in the inhibition of cell growth in several representative acute leukemia cell lines and was much more potent than CBP/p300 bromodomain and catalytic domain inhibitors. JET-209 effectively inhibited tumor growth in xenograft tumor models at tolerated dose schedules. JET-209 is a promising lead compound for further evaluation and optimization toward the development of a CBP/p300 degrader for the treatment of human cancers.

Discovery of ARD-2051 as a Potent and Orally Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.

CoGT: Ensemble Machine Learning Method and Its Application on JAK Inhibitor Discovery.

The discovery of new drug candidates to inhibit an intended target is a complex and resource-consuming process. A machine learning (ML) method for predicting drug-target interactions (DTI) is a potential solution to improve the efficiency. However, traditional ML approaches have limitations in accuracy. In this study, we developed a novel ensemble model CoGT for DTI prediction using multilayer perceptron (MLP), which integrated graph-based models to extract non-Euclidean molecular structures and large pretrained models, specifically chemBERTa, to process simplified molecular input line entry systems (SMILES). The performance of CoGT was evaluated using compounds inhibiting four Janus kinases (JAKs). Results showed that the large pretrained model, chemBERTa, was better than other conventional ML models in predicting DTI across multiple evaluation metrics, while the graph neural network (GNN) was effective for prediction on imbalanced data sets. To take full advantage of the strengths of these different models, we developed an ensemble model, CoGT, which outperformed other individual ML models in predicting compounds' inhibition on different isoforms of JAKs. Our data suggest that the ensemble model CoGT has the potential to accelerate the process of drug discovery.

DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates.

Nonalcoholic steatohepatitis (NASH) prevalence is rising with no pharmacotherapy approved. A major hurdle in NASH drug development is the poor translatability of preclinical studies to safe/effective clinical outcomes, and recent failures highlight a need to identify new targetable pathways. Dysregulated glycine metabolism has emerged as a causative factor and therapeutic target in NASH. Here, we report that the tripeptide DT-109 (Gly-Gly-Leu) dose-dependently attenuates steatohepatitis and fibrosis in mice. To enhance the probability of successful translation, we developed a nonhuman primate model that histologically and transcriptionally mimics human NASH. Applying a multiomics approach combining transcriptomics, proteomics, metabolomics, and metagenomics, we found that DT-109 reverses hepatic steatosis and prevents fibrosis progression in nonhuman primates, not only by stimulating fatty acid degradation and glutathione formation, as found in mice, but also by modulating microbial bile acid metabolism. Our studies describe a highly translatable NASH model and highlight the need for clinical evaluation of DT-109.

A selective small-molecule STAT5 PROTAC degrader capable of achieving tumor regression in vivo.

Signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target, but successful targeting of STAT5 has proved to be difficult. Here we report the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and more than 6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and the therapeutic potential of selective STAT5 protein depletion and inhibition but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.

Discovery of a Potent and Selective STAT5 PROTAC Degrader with Strong Antitumor Activity In Vivo in Acute Myeloid Leukemia.

STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.

ASGARD is A Single-cell Guided Pipeline to Aid Repurposing of Drugs.

Single-cell RNA sequencing technology has enabled in-depth analysis of intercellular heterogeneity in various diseases. However, its full potential for precision medicine has yet to be reached. Towards this, we propose A Single-cell Guided Pipeline to Aid Repurposing of Drugs (ASGARD) that defines a drug score to recommend drugs by considering all cell clusters to address the intercellular heterogeneity within each patient. ASGARD shows significantly better average accuracy on single-drug therapy compared to two bulk-cell-based drug repurposing methods. We also demonstrated that it performs considerably better than other cell cluster-level predicting methods. In addition, we validate ASGARD using the drug response prediction method TRANSACT with Triple-Negative-Breast-Cancer patient samples. We find that many top-ranked drugs are either approved by the Food and Drug Administration or in clinical trials treating corresponding diseases. In conclusion, ASGARD is a promising drug repurposing recommendation tool guided by single-cell RNA-seq for personalized medicine. ASGARD is free for educational use at https://github.com/lanagarmire/ASGARD .

Design and Synthesis of Orally Active Quinolyl Pyrazinamides as Sigma 2 Receptor Ligands for the Treatment of Pancreatic Cancer.

Sigma 2 receptor (σ2R) is overexpressed in select cancers and is regarded as a biomarker for tumor proliferation. σ2R ligands are emerging as promising theranostics for cancer and neurodegenerative diseases. Herein, we describe the design and synthesis of a series of novel quinolyl pyrazinamides as selective and potent σ2R ligands that show sub-micromolar potency in pancreatic cancer cell lines. Compounds 14 (JR1-157) and 17 (JR2-298) bind σ2R with Ki of 47 and 10 nM, respectively. Importantly, compound 14 has an oral bioavailability of 60% and shows significant in vivo efficacy without obvious toxicity in a syngeneic model of pancreatic cancer. The cytotoxicity of the quinolyl pyrazinamides significantly enhanced in the presence of copper and diminished in the presence of the copper-chelator tetrathiomolybdate. In conclusion, compound 14 is water-soluble, metabolically stable, orally active, and increases the expression of the autophagy marker LC3B and warrants further development for the treatment of pancreatic cancer.

Regulatory utility of physiologically-based pharmacokinetic modeling to support alternative bioequivalence approaches and risk assessment: A workshop summary report.

This report summarizes the proceedings for day 2 sessions 1 and 3 of the 2-day public workshop entitled "Regulatory Utility of Mechanistic Modeling to Support Alternative Bioequivalence Approaches," a jointly sponsored workshop by the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics (CRCG). The aims of this workshop were: (1) to discuss how mechanistic modeling, including physiologically-based pharmacokinetic (PBPK) modeling and simulation, can support product development, and regulatory submissions; (2) to share the current state of mechanistic modeling for bioequivalence (BE) assessment through case studies; (3) to establish a consensus on best practices for using PBPK modeling for BE assessment to help drive further investment by the generic drug industry into mechanistic modeling and simulation; and (4) to introduce the concept of a Model Master File to improve model-sharing. The theme of day 2 covered PBPK absorption model for oral products as an alternative BE approach and a tool for supporting risk assessment and biowaiver (session 1), oral PBPK for evaluating the impact of food on BE (session 2), successful cases, and challenges for oral PBPK (session 3). This report summarizes the topics of the presentations of day 2 sessions 1 and session 3 from FDA, academia, and pharmaceutical industry, including the current status of oral PBPK, case examples as well as the challenges and opportunities in this area. In addition, panel discussions on the utility of oral PBPK in both new drugs and generic drugs from regulatory and industry perspective are also summarized.