Small molecule immuno-oncology therapeutic agents

Treatment of cancer by activation of an antitumor immune response is now a widely practiced and well-accepted approach to therapy. However, despite dramatic responses in some patients, the high proportion of unresponsive patients points to a considerable unmet medical need. Although antibody therapies have led the way, small molecule immuno-oncology agents are close behind. This perspective provides an overview of some of the many small molecule approaches being explored. It encompasses small molecule modulators of validated targets such as programed cell death 1 (PD-1) as well as novel approaches still to be proven clinically.     

FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo

Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas.     

RNA as a small molecule druggable target

Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents.     

Yeast "N"-hybrid systems for protein-protein and drug-protein interaction discovery

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets.     

Therapeutic peptides for CNS indications: Progress and challenges

Attacking neurodegeneration and promoting neuroprotection have been the holy grail in neurology for almost 20 years and represent an area of high unmet medical need. However, indications like Alzheimer’s disease and stroke are areas in drug development fraught with failure. This review will highlight three CNS peptide programs which are tackling targets and indications in which traditional small molecule approaches have been difficult and challenging. The targets for these potential peptide therapeutics include the NMDA receptor, γ-secretase, and cyclin-dependent kinase in which direct inhibition has resulted in on-target (not compound related) problems. For example, direct inhibition of γ-secretase has resulted in gastrointestinal abnormalities and inhibition of the NMDA receptor can result in hallucinations, dizziness, out-of-body sensations, and nightmares. When confronted with show-stopping side effects, the CNS peptide programs profiled in this review strike the problem with intervention and disruption of selective protein-protein interactions. The goal of these peptide programs is to produce selective therapeutics with a better safety profile.     

Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.     

Discovery of novel regulators of aldehyde dehydrogenase isoenzymes

Over the past three years we have been involved in high-throughput screening in an effort to discover novel small molecular modulators of aldehyde dehydrogenase (ALDH) activity. In particular, we have been interested in both the activation and inhibition of the three commonly studied isoenzymes, ALDH1A1, ALDH2 and ALDH3A1, as their distinct, yet overlapping substrate specificities, present a particularly difficult challenge for inhibitor discovery and design. Activation of ALDH2 has been shown to benefit cardiovascular outcome following periods of ischemia and renewed interest in specific inhibition of ALDH2 has application for alcohol aversion therapy, and more recently, in cocaine addiction. In contrast, inhibition of either ALDH1A1 or ALDH3A1 has application in cancer treatments where the isoenzymes are commonly over-expressed and serve as markers for cancer stem cells. We are taking two distinct approaches for these screens: in vitro enzyme activity screens using chemical libraries and virtual computational screens using the structures of the target enzymes as filters for identifying potential inhibitors, followed by in vitro testing of their ability to inhibit their intended targets. We have identified selective inhibitors of each of these three isoenzymes with inhibition constants in the high nanomolar to low micromolar range from these screening procedures. Together, these inhibitors provide proof for concept that selective inhibition of these broad specificity general detoxication enzymes through small molecule discovery and design is possible.     

AlphaFold2 protein structure prediction: Implications for drug discovery

The drug discovery process involves designing compounds to selectively interact with their targets. The majority of therapeutic targets for low molecular weight (small molecule) drugs are proteins. The outstanding accuracy with which recent artificial intelligence methods compile the three-dimensional structure of proteins has made protein targets more accessible to the drug design process. Here, we present our perspective of the significance of accurate protein structure prediction on various stages of the small molecule drug discovery life cycle focusing on current capabilities and assessing how further evolution of such predictive procedures can have a more decisive impact in the discovery of new medicines.     

Development of HIV entry inhibitors targeted to the coiled-coil regions of gp41

The discoveries that synthetic peptides corresponding to the N- and C-terminal heptad repeat (HR) regions of gp41 have potent anti-HIV activity opened a new avenue to identification of small molecule HIV entry inhibitors targeted to the HIV gp41 coiled-coil regions. Based on the structural information of the HIV gp41 core, three distinct approaches to develop small molecule anti-HIV agents have been reported. Each of these approaches has specific advantages, which will have complementary effects on the design of new strategies for identification of more potent HIV entry inhibitors. It is expected that novel antiviral drugs targeted to the HIV gp41 coiled-coil regions will be developed in the near future for the chemotherapy and/or prophylaxis of HIV infection and AIDS.     

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S.?aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S.?aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S?aureus. These are generally small molecules that inhibit virulence targets in S.?aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.     

Solvent-induced proteome profiling for proteomic quantitation and target discovery of small molecular drugs

Target identification by modification-free proteomic approaches can potentially reveal the pharmacological mechanism of small molecular compounds. By combining the recent solvent-induced protein precipitation (SIP) method with TMT-labeling quantitative proteomics, we propose solvent-induced proteome profiling (SIPP) approach to identify small molecule–protein interactions. The SIPP approach enables to depict denaturation curves of the target protein by varying concentrations of organic solvents to induce unfolding and precipitation of the cellular proteome. By using this approach, we have successfully identified the known targets of market drugs and natural products and extended the proteome information of SIP for target identification.     

Morphoproteomics: exposing protein circuitries in tumors to identify potential therapeutic targets in cancer patients

Morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering molecular targets amenable to specific intervention, thereby customizing therapy for individual patients. This review considers the clinical application of morphoproteomics in malignant cells in the context of currently available pharmaceutical agents and discusses opportunities for combinatorial approaches that involve one or more small molecule inhibitors and single-agent chemotherapy with relatively low toxicity profiles. Future directions that involve focusing on points of convergence in signal transduction pathways and which integrate morphoproteomic with genomic and pharmacoproteomic and protein-function microarray data are offered.     

Morphoproteomics: exposing protein circuitries in tumors to identify potential therapeutic targets in cancer patients

Morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering molecular targets amenable to specific intervention, thereby customizing therapy for individual patients. This review considers the clinical application of morphoproteomics in malignant cells in the context of currently available pharmaceutical agents and discusses opportunities for combinatorial approaches that involve one or more small molecule inhibitors and single-agent chemotherapy with relatively low toxicity profiles. Future directions that involve focusing on points of convergence in signal transduction pathways and which integrate morphoproteomic with genomic and pharmacoproteomic and protein-function microarray data are offered.     

Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.     

Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson''s disease), auditory disorders (Waardenburg''s syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.     

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.     

Protein kinases as targets for anti-parasitic chemotherapy

Parasitic protozoa infecting humans have a staggering impact on public health, especially in the developing world. Furthermore, several protozoan species are major pathogens of domestic animals and have a considerable impact on food production. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in protein kinases inhibitors as potential drugs against a variety of diseases, the possibility that protein kinases may represent targets for novel anti-parasitic agents is being explored. Research into parasite protein kinases has benefited greatly from genome and EST sequencing projects, with the genomes of a few species fully sequenced (notably that of the human malaria parasite Plasmodium falciparum) and several more under way. The overall picture that emerged from research in this area shows that the phylogenetic isolation of parasitic protozoa is reflected by atypical structural and functional properties of many of their protein kinase homologues. Likewise, evidence is emerging, which suggests that the organisation of some otherwise well-conserved signal transduction pathways is divergent in some parasitic species. The differences between protein kinases of a parasite and their homologues in its host cell suggest that specific inhibition of the former can be achieved. The development of anti-parasitic drugs based on protein kinase inhibition is being pursued following two avenues: one consists of screening chemical libraries on recombinant enzymes; several protein kinases from parasitic protozoa are now available for this approach. The second approach relies on the identification of the molecular targets of kinase inhibitors which display anti-parasitic properties. This has led to promising developments in a few instances, in particular regarding PKG as a drug target against Eimeria and Toxoplasma, and purvalanol B, a purine-based CDK inhibitor which appears to affect unexpected targets in several protozoan parasites. The recent resolution of the structure of a Plasmodium protein kinase complexed with small inhibitory molecules opens the way to a rational approach towards the design of anti-parasitic drugs based on kinase inhibition.     

A new era for small molecule screening: from new targets, such as JAK2 V617F, to complex cellular screens

Traditionally reserved to research and development in pharmaceutical companies, screening of small molecule libraries is rapidly becoming an approach undertaken by academic laboratories. Novel cellular assays, sensitive systems to probe function, emerging new molecular targets are just some of the reasons explaining this shift. Targets of small molecules identified in cellular screens begin to be amenable to identification by elegant genetic approaches, such as probing toxicity of candidate small molecules on libraries of genetically modified yeast strains. Several new targets, such as JAK2 V617F, an activated JAK2 (Janus Kinase 2) mutant genetically associated with the majority of human myeloproliferative neoplasms, are being actively pursued. In this Review Series, we will learn how libraries of small molecules are harnessed to identify novel molecules, that alone or in combination, have the ability to alter cell fate, cell signalling, gene expression or response to extracellular cues.     

Targeting Focal Adhesion Kinase Suppresses the Malignant Phenotype in Rhabdomyosarcoma Cells

Despite the tremendous advances in the treatment of childhood solid tumors, rhabdomyosarcoma (RMS) continues to provide a therapeutic challenge. Children with metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumorigenesis. Signaling pathways both upstream and downstream to FAK have been found to be important in sarcoma tumorigenesis, leading us to hypothesize that FAK would be present in RMS and would impact cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric alveolar and embryonal RMS tumor specimens and cell lines. We also examined the effects of FAK inhibition upon two RMS cell lines utilizing parallel approaches including RNAi and small molecule inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Furthermore, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse RMS xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in RMS and may provide desperately needed novel therapeutic strategies for these difficult-to-treat tumors.