Development of VPC-70619, a Small-Molecule N-Myc Inhibitor as a Potential Therapy for Neuroendocrine Prostate Cancer.

The Myc family of transcription factors are involved in the development and progression of numerous cancers, including prostate cancer (PCa). Under the pressure of androgen receptor (AR)-directed therapies resistance can occur, leading to the lethal form of PCa known as neuroendocrine prostate cancer (NEPC), characterized among other features by N-Myc overexpression. There are no clinically approved treatments for NEPC, translating into poor patient prognosis and survival. Therefore, there is a pressing need to develop novel therapeutic avenues to treat NEPC patients. In this study, we investigate the N-Myc-Max DNA binding domain (DBD) as a potential target for small molecule inhibitors and utilize computer-aided drug design (CADD) approaches to discover prospective hits. Through further exploration and optimization, a compound, VPC-70619, was identified with notable anti-N-Myc potency and strong antiproliferative activity against numerous N-Myc expressing cell lines, including those representing NEPC.

Comprehensive Consensus Analysis of SARS-CoV-2 Drug Repurposing Campaigns.

The current COVID-19 pandemic has elicited extensive repurposing efforts (both small and large scale) to rapidly identify COVID-19 treatments among approved drugs. Herein, we provide a literature review of large-scale SARS-CoV-2 antiviral drug repurposing efforts and highlight a marked lack of consistent potency reporting. This variability indicates the importance of standardizing best practices-including the use of relevant cell lines, viral isolates, and validated screening protocols. We further surveyed available biochemical and virtual screening studies against SARS-CoV-2 targets (Spike, ACE2, RdRp, PLpro, and Mpro) and discuss repurposing candidates exhibiting consistent activity across diverse, triaging assays and predictive models. Moreover, we examine repurposed drugs and their efficacy against COVID-19 and the outcomes of representative repurposed drugs in clinical trials. Finally, we propose a drug repurposing pipeline to encourage the implementation of standard methods to fast-track the discovery of candidates and to ensure reproducible results.

Quantitative Structure-Price Relationship (QS$R) Modeling and the Development of Economically Feasible Drug Discovery Projects.

In recent years, the field of quantitative structure-activity/property relationship (QSAR/QSPR) modeling has developed into a stable technology capable of reliably predicting new bioactive molecules. With the availability of inexpensive commercial sources of both synthetic chemicals and bioactivity assays, a cheminformatics-savvy scientist can readily establish a virtual drug discovery enterprise. A skilled computational chemist can not only develop a computer-aided drug discovery pipeline but also acquire or have the drug candidates made inexpensively for economical screening of desired on-target activity, critical off-target effects, and essential drug-likeness properties. As part of our drug discovery pipeline, a novel machine-learning model was built to relate chemical structures of synthetically accessible molecules to their prices. The model was trained from our "in stock" and "made on demand" diverse chemical entities, ranging in price from $20 to >$10,000. This novel model is encoded here as the quantitative structure-price relationship (QS$R) model.

Automated discovery of noncovalent inhibitors of SARS-CoV-2 main protease by consensus Deep Docking of 40 billion small molecules.

Recent explosive growth of 'make-on-demand' chemical libraries brought unprecedented opportunities but also significant challenges to the field of computer-aided drug discovery. To address this expansion of the accessible chemical universe, molecular docking needs to accurately rank billions of chemical structures, calling for the development of automated hit-selecting protocols to minimize human intervention and error. Herein, we report the development of an artificial intelligence-driven virtual screening pipeline that utilizes Deep Docking with Autodock GPU, Glide SP, FRED, ICM and QuickVina2 programs to screen 40 billion molecules against SARS-CoV-2 main protease (Mpro). This campaign returned a significant number of experimentally confirmed inhibitors of Mpro enzyme, and also enabled to benchmark the performance of twenty-eight hit-selecting strategies of various degrees of stringency and automation. These findings provide new starting scaffolds for hit-to-lead optimization campaigns against Mpro and encourage the development of fully automated end-to-end drug discovery protocols integrating machine learning and human expertise.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy.

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.

Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells.

OBJECTIVE: The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells. MATERIALS AND METHODS: A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents. RESULTS: Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor. CONCLUSION: To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.

Mammalian artificial chromosomes and clinical applications for genetic modification of stem cells: an overview.

Modifying multipotent, self-renewing human stem cells with mammalian artificial chromosomes (MACs), present a promising clinical strategy for numerous diseases, especially ex vivo cell therapies that can benefit from constitutive or overexpression of therapeutic gene(s). MACs are nonintegrating, autonomously replicating, with the capacity to carry large cDNA or genomic sequences, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression, and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in progenitor cells. The status quo is that the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells. We will describe the progress of MAC technologies, the subsequent modifications of stem cells, and discuss the establishment of MAC platform stem cell lines to facilitate proof-of-principle studies and preclinical development.

Development of mammalian artificial chromosomes for the treatment of genetic diseases: Sandhoff and Krabbe diseases.

Mammalian artificial chromosomes (MACs) are being developed as alternatives to viral vectors for gene therapy applications, as they allow for the introduction of large payloads of genetic information in a non-integrating, autonomously replicating format. One class of MACs, the satellite DNA-based artificial chromosome expression vehicle (ACE), is uniquely suited for gene therapy applications, in that it can be generated denovo in cells, along with being easily purified and readily transferred into a variety of recipient cell lines and primary cells. To facilitate the rapid engineering of ACEs, the ACE System was developed, permitting the efficient and reproducible loading of pre-existing ACEs with DNA sequences and/or target gene(s). As a result, the ACE System and ACEs are unique and versatile platforms for ex vivo gene therapy strategies that circumvent and alleviate existing safety and delivery limitations surrounding conventional gene therapy vectors. This review will focus on the status of MAC technologies and, in particular, the application of the ACE System towards an ex vivo gene therapy treatment of lysosomal storage diseases, specifically Sandhoff (MIM #268800) and Krabbe (MIM #245200) diseases.