Small Molecule Enzyme Inhibitors Research Articles

Metabolic modeling predicts unique drug targets in Borrelia burgdorferi.

Lyme disease is often treated using long courses of antibiotics, which can cause side effects for patients and risks the evolution of antimicrobial resistance. Narrow-spectrum antimicrobials would reduce these risks, but their development has been slow because the Lyme disease bacterium, Borrelia burgdorferi, is difficult to work with in the laboratory. To accelerate the drug discovery pipeline, we developed a computational model of B. burgdorferi's metabolism and used it to predict essential enzymatic reactions whose inhibition prevented growth in silico. These predictions were validated using small-molecule enzyme inhibitors, several of which were shown to have specific activity against B. burgdorferi. Although the specific compounds used are not suitable for clinical use, we aim to use them as lead compounds to develop optimized drugs targeting the pathways discovered here.

Quantitative Biochemical Analysis of Deadenylase Enzymes Using Fluorescence and Chemiluminescence-Based Assays.

Deadenylase enzymes play a key role in mRNA degradation and RNA processing. In this chapter, we describe two activity assays for the quantitative biochemical analysis of deadenylase enzymes, which can easily be adapted for other nuclease enzymes. The assays use distinct principles of detection, which are based on differential annealing of a probe complementary to the substrate RNA or detection of adenosine monophosphate (AMP). The assays are sensitive, flexible, and can be used in low-throughput tube-based formats and 96-well or 384-well plate formats. The assays rely on plate reader detection and can be carried out using manual pipetting or robotic liquid handling equipment. In addition to two activity assays, we describe differential scanning fluorimetry (thermal shift assay) as a complementary assay that allows the direct characterization of ligand binding to deadenylase enzymes. The assays can be useful for the characterization of deadenylase variants and are particularly suitable for the discovery and development of small-molecule inhibitors of deadenylase enzymes.

Development of an online UPLC-PDA-ESI-Q-TOF-MS-LOX-FLD system for rapid screening of anti-inflammatory compounds in Polygala tenuifolia Willd

In this study, the first ultra-high performance liquid chromatography-photo-diode array-electrospray ionization-quadrupole-time-of-flight-mass spectrometry-lipoxygenase-fluorescence detector (UPLC-PDA-ESI-Q-TOF-MS-LOX-FLD) online system was developed for the identification and evaluation of anti-inflammatory active ingredients in Polygala tenuifolia Willd. Using this system, the UPLC fingerprints, mass fragments and LOX-binding peak profiles in the samples were rapidly and simultaneously obtained. A total of 101 compounds were isolated and identified and 38 compounds (11 oligosaccharide esters, nine xanthones, 17 saponins, and one glycosyloxyflavone) showed strong LOX-binding activity. Six compounds were selected to study their LOX-binding ability, and the results indicated that the content of the six compounds had a good linear relationship with the LOX-binding ability, and it was found that the substitution position, the type of substituent and the number of glycosyl groups all had a certain influence on the LOX-binding ability of the compounds. The LOX-binding activities of 10 compounds were verified by the surface plasmon resonance (SPR) technique and the activity results were consistent with the online system. After validation, we identified 7 active compounds that combined with LOX to exert anti-inflammatory effects for the first time. All the results fully demonstrate the efficiency, stability and reliability of the online system and this work provides an exemplary and useful method for the rapid screening of potential anti-inflammatory active compounds in P. tenuifolia and other traditional Chinese medicines. At the same time, it provides a new direction for screening small molecule inhibitors of enzymes like LOX.

Dose Adjustment of Poly (ADP‑Ribose) Polymerase Inhibitors in Patients with Hepatic or Renal Impairment.

Poly (ADP-ribose) polymerase (PARP) inhibitors are small-molecule inhibitors of PARP enzymes (including PARP1, PARP2, and PARP3) that exhibit activity against tumor cells with defects in DNA repair. In recent years, five PARP inhibitors, olaparib, niraparib, rucaparib, talazoparib and veliparib, have been developed for the treatment of solid tumors, particularly in patients with breast-related cancer antigen (BRCA) 1/2 mutations, or those without a functional homologous recombination repair pathway. These novel treatments exhibit improved efficacy and toxicity when compared to conventional chemotherapy agents. The five PARP inhibitors are eliminated primarily via the liver and kidneys, hepatic or renal impairment may significantly affect their pharmacokinetics (PK). Therefore, it is important to know the effects of hepatic or renal impairment on the PK and safety of PARP inhibitors. In this review, we characterize and summarize the effects of hepatic and renal function on the PK of PARP inhibitors and provide specific recommendations for clinicians when prescribing PARP inhibitors in patients with hepatic or renal impairment.

Efficacy and Limitations of Chemically Diverse Small-Molecule Enzyme-Inhibitors against the Synergistic Coagulotoxic Activities of Bitis Viper Venoms.

Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have begun to focus on the use of small-molecule inhibitors as potential candidates for the neutralisation of key snake venom toxins and as potential field therapies. Bitis vipers represent some of the most medically important as well as frequently encountered snake species in Africa, with a number of species possessing anticoagulant phospholipase A2 (PLA2) toxins that prevent the prothrombinase complex from inducing clot formation. Additionally, species within the genus are known to exert pseudo-procoagulant activity, whereby kallikrein enzymatic toxins cleave fibrinogen to form a weak fibrin clot that rapidly degrades, thereby depleting fibrinogen levels and contributing to the net anticoagulant state. Utilising well-validated coagulation assays measuring time until clot formation, this study addresses the in vitro efficacy of three small molecule enzyme inhibitors (marimastat, prinomastat and varespladib) in neutralising these aforementioned activities. The PLA2 inhibitor varespladib showed the greatest efficacy for the neutralisation of PLA2-driven anticoagulant venom activity, with the metalloproteinase inhibitors prinomastat and marimastat both showing low and highly variable degrees of cross-neutralisation with PLA2 anticoagulant toxicity. However, none of the inhibitors showed efficacy in neutralising the pseudo-procoagulant venom activity exerted by the venom of B. caudalis. Our results highlight the complex nature of snake venoms, for which single-compound treatments will not be universally effective, but combinations might prove highly effective. Despite the limitations of these inhibitors with regards to in vitro kallikrein enzyme pseudo-procoagulant venom activity, our results further support the growing body of literature indicating the potential use of small molecule inhibitors to enhance first-aid treatment of snakebite envenoming, particularly in cases where hospital and thus antivenom treatment is either unavailable or far away.

Function of m6A and its regulation of domesticated animals' complex traits.

N6-methyladenosine (m6A) is the most functionally important epigenetic modification in RNA. The m6A modification widely exists in mRNA and noncoding RNA, influences the mRNA processing, and regulates the secondary structure and maturation of noncoding RNA. Studies showed the important regulatory roles of m6A modification in animal's complex traits, such as development, immunity, and reproduction-related traits. As an important intermediate stage from animal genome to phenotype, the function of m6A in the complex trait formation of domestic animals cannot be neglected. This review discusses recent research advances on m6A modification in well-studied organisms, such as human and model organisms, and introduces m6A detection technologies, small-molecule inhibitors of m6A-related enzymes, interaction between m6A and other biological progresses, and the regulation mechanisms of m6A in domesticated animals' complex traits.

Kynurenine Pathway Metabolites as Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that deteriorates cognitive function. Patients with AD generally exhibit neuroinflammation, elevated beta-amyloid (Aβ), tau phosphorylation (p-tau), and other pathological changes in the brain. The kynurenine pathway (KP) and several of its metabolites, especially quinolinic acid (QA), are considered to be involved in the neuropathogenesis of AD. The important metabolites and key enzymes show significant importance in neuroinflammation and AD. Meanwhile, the discovery of changed levels of KP metabolites in patients with AD suggests that KP metabolites may have a prominent role in the pathogenesis of AD. Further, some KP metabolites exhibit other effects on the brain, such as oxidative stress regulation and neurotoxicity. Both analogs of the neuroprotective and antineuroinflammation metabolites and small molecule enzyme inhibitors preventing the formation of neurotoxic and neuroinflammation compounds may have potential therapeutic significance. This review focused on the KP metabolites through the relationship of neuroinflammation in AD, significant KP metabolites, and associated molecular mechanisms as well as the utility of these metabolites as biomarkers and therapeutic targets for AD. The objective is to provide references to find biomarkers and therapeutic targets for patients with AD.

A Review of Small-Molecule Inhibitors of One-Carbon Enzymes: SHMT2 and MTHFD2 in the Spotlight.

Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.

Small Molecule-Based Enzyme Inhibitors in the Treatment of Primary Hyperoxalurias.

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.

Structural Studies on the Inhibitory Binding Mode of Aromatic Coumarinic Esters to Human Kallikrein-Related Peptidase 7.

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.

Therapeutics-how to treat phase separation-associated diseases.

Liquid–liquid phase separation has drawn attention as many neurodegeneration or cancer-associated proteins are able to form liquid membraneless compartments (condensates) by liquid–liquid phase separation. Furthermore, there is rapidly growing evidence that disease-associated mutation or post-translational modification of these proteins causes aberrant location, composition or physical properties of the condensates. It is ambiguous whether aberrant condensates are always causative in disease mechanisms, however they are likely promising potential targets for therapeutics. The conceptual framework of liquid–liquid phase separation provides opportunities for novel therapeutic approaches. This review summarises how the extensive recent advances in understanding control of nucleation, growth and composition of condensates by protein post-translational modification has revealed many possibilities for intervention by conventional small molecule enzyme inhibitors. This includes the first proof-of-concept examples. However, understanding membraneless organelle formation as a physical chemistry process also highlights possible physicochemical mechanisms of intervention. There is huge demand for innovation in drug development, especially for challenging diseases of old age including neurodegeneration and cancer. The conceptual framework of liquid–liquid phase separation provides a new paradigm for thinking about modulating protein function and is very different from enzyme lock-and-key or structured binding site concepts and presents new opportunities for innovation.

Metabolomics Analysis Identifies Sphingolipids as Key Signaling Moieties in Appressorium Morphogenesis and Function in Magnaporthe oryzae.

The blast fungus initiates infection using a heavily melanized, dome-shaped infection structure known as the appressorium, which forcibly ruptures the cuticle to enter the rice leaf tissue. How this process takes place remains not fully understood. Here, we used untargeted metabolomics analyses to profile the metabolome of developing appressoria and identified significant changes in six key metabolic pathways, including early sphingolipid biosynthesis. Analyses employing small molecule inhibitors, gene disruption, or genetic and chemical complementation demonstrated that ceramide compounds of the sphingolipid biosynthesis pathway are essential for normal appressorial development controlled by mitosis. In addition, ceramide was found to act upstream from the protein kinase C-mediated cell wall integrity pathway during appressorium repolarization and pathogenicity in rice blast. Further discovery of the sphingolipid biosynthesis pathway revealed that glucosylceramide (GlcCer) synthesized by ceramide is the key substance affecting the pathogenicity of Magnaporthe oryzae Our results provide new insights into the chemical moieties involved in the infection-related signaling networks, thereby revealing a potential target for the development of novel control agents against the major disease of rice and other cereals.IMPORTANCE Our untargeted analysis of metabolomics throughout the course of pathogenic development gave us an unprecedented high-resolution view of major shifts in metabolism that occur in the topmost fungal pathogen that infects rice, wheat, barley, and millet. Guided by these metabolic insights, we demonstrated their practical application by using two different small-molecule inhibitors of sphingolipid biosynthesis enzymes to successfully block the pathogenicity of M. oryzae Our study thus defines the sphingolipid biosynthesis pathway as a key step and potential target that can be exploited for the development of antifungal agents. Furthermore, future investigations that exploit such important metabolic intermediates will further deepen our basic understanding of the molecular mechanisms underlying the establishment of fungal blast disease in important cereal crops.

Substrate-Selective Enzyme Inhibitors

Enzymes with multiple substrates pose a unique challenge for drug development because of an increased potential for on-target side effects. Maianti and colleagues (Nat. Chem. Biol., 2019) identify novel exo-site inhibitors with abilities to alter the substrate-selectivity of insulin-degrading enzymes (IDE). Their work illuminates new therapeutic avenues for discovering small-molecule enzyme inhibitors and redefines our current understanding of drugging enzymes with multiple substrates.

Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

Biosynthetic Oligoclonal Antivenom (BOA) for Snakebite and Next-Generation Treatments for Snakebite Victims.

Snakebite envenoming is a neglected tropical disease that each year claims the lives of 80,000–140,000 victims worldwide. The only effective treatment against envenoming involves intravenous administration of antivenoms that comprise antibodies that have been isolated from the plasma of immunized animals, typically horses. The drawbacks of such conventional horse-derived antivenoms include their propensity for causing allergenic adverse reactions due to their heterologous and foreign nature, an inability to effectively neutralize toxins in distal tissue, a low content of toxin-neutralizing antibodies, and a complex manufacturing process that is dependent on husbandry and procurement of snake venoms. In recent years, an opportunity to develop a fundamentally novel type of antivenom has presented itself. By using modern antibody discovery strategies, such as phage display selection, and repurposing small molecule enzyme inhibitors, next-generation antivenoms that obviate the drawbacks of existing plasma-derived antivenoms could be developed. This article describes the conceptualization of a novel therapeutic development strategy for biosynthetic oligoclonal antivenom (BOA) for snakebites based on recombinantly expressed oligoclonal mixtures of human monoclonal antibodies, possibly combined with repurposed small molecule enzyme inhibitors.

Vascular Adhesion Protein-1: A Cell Surface Amine Oxidase in Translation.

Significance: Vascular adhesion protein-1 (VAP-1) is an ectoenzyme that oxidates primary amines in a reaction producing also hydrogen peroxide. VAP-1 on the blood vessel endothelium regulates leukocyte extravasation from the blood into tissues under physiological and pathological conditions.Recent Advances: Inhibition of VAP-1 by neutralizing antibodies and by several novel small-molecule enzyme inhibitors interferes with leukocyte trafficking and alleviates inflammation in many experimental models. Targeting of VAP-1 also shows beneficial effects in several other diseases, such as ischemia/reperfusion, fibrosis, and cancer. Moreover, soluble VAP-1 levels may serve as a new prognostic biomarker in selected diseases.Critical Issues: Understanding the contribution of the enzyme activity-independent and enzyme activity-dependent functions, which often appear to be mediated by the hydrogen peroxide production, in the VAP-1 biology will be crucial. Similarly, there is a pressing need to understand which of the VAP-1 functions are regulated through the modulation of leukocyte trafficking, and what is the role of VAP-1 synthesized in adipose and smooth muscle cells.Future Directions: The specificity and selectivity of new VAP-1 inhibitors, and their value in animal models under therapeutic settings need to be addressed. Results from several programs studying the therapeutic potential of VAP-1 inhibition, which now are in clinical trials, will reveal the relevance of this amine oxidase in humans.

Abstract 1949: A compound screen reveals potential novel therapeutic targets for chordoma: Metabolic stress response and epigenetic control of brachyury

Abstract Chordoma is a rare bone cancer, showing notochordal differentiation, that develops in axial skeleton in adults and children. Patients have a median survival of 7 years and radical surgical resection is the main treatment for this morbid disease, which does not respond to cytotoxic chemotherapy. We have previously demonstrated that EGFR inhibitors represent the almost unique family of kinase inhibitors to exert an effect on chordoma cell lines proliferation. However not all cell lines respond to these agents and drug resistance is likely to occur. Genomic studies have revealed that chordomas do not harbor recurrent alterations in kinases whereas chromatin-remodelling genes are altered in at least 20% of cases. The transcription factor brachyury (T) is the diagnostic hallmark of chordoma and is strongly implicated in its pathogenesis. T is regulated during embryonic development at the epigenetic level, suggesting that epigenetic inhibitors may represent a novel therapeutic approach for this disease. In this study, we have undertaken a medium throughput focused compound screen (n=91) using validated small molecule inhibitors of enzymes involved in chromatin biology and metabolic pathways. The alamar blue assay was employed to assess cell viability. Screening revealed activity in a number of compounds targeting the jumonji domain-containing lysine demethylases, including GSK-J4 and KDOBA67, two structurally closely related compounds that mainly target KDM6A (aka UTX) and KMD6B (JMJD3). These compounds were effective in all five chordoma cell lines (UCH1, UCH2, MUG-Chor, UM-Chor, UCH7) tested. In contrast to EGFR inhibitors, these compounds induced downregulation of T at the transcriptional and protein level. Preliminary results suggest this is achieved via the induction of a metabolic stress response as well as through the epigenetic regulation of T, the latter being brought by increased levels of H3K27me3. We also found that Halofuginone, a highly specific inhibitor of the enzyme glutamyl-prolyl tRNA synthetase already tested in phase I autoimmunity clinical trials, induced a metabolic stress response, similar to KDM6 inhibitors, in all chordoma cell lines. Moreover, Halofuginone treatment of a chordoma PDX model demonstrated 44% tumor growth inhibition (p=0.0052). In conclusion, we have identified epigenetic and metabolic pathways that represent potential novel targets for the treatment of chordoma. Citation Format: Lucia Cottone, Edward Hookway, Graham Wells, Lorena Ligammari, Patrick Lombard, Ralph Mazitschek, Josh Sommer, Udo Oppermann, Adrienne M. Flanagan. A compound screen reveals potential novel therapeutic targets for chordoma: Metabolic stress response and epigenetic control of brachyury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1949.

Abstract 3510: Small molecule ACSS2 inhibitors target acetate metabolizing tumor cells in hypoxic conditions

Abstract BACKGROUND Tumors such as glioblastoma proliferating under hypoxic, hypoglycemic and hypolipidemic conditions derive almost half of their nutritional requirement from the short chain fatty acid acetate. Acetyl CoA synthetases (ACSS 1 to 3) are a family of enzymes that metabolize acetate to the multifunctional metabolite acetyl-CoA. Acetyl-coA is a key precursor of cellular biomass and is required for epigenetic modification. There is an increasing body of clinical evidence that places acetate and ACSS2 at a critical metabolic node in tumors. We have developed potent small molecule enzyme inhibitors of ACSS2 that block the functional activity of the enzyme in vitro and in vivo. Furthermore, these inhibitors synergize with known chemotherapeutics to eliminate cancer cells in hypoxic conditions. METHODS A pharmacophore based medicinal chemistry campaign yielded initial hits which were refined in subsequent rounds of structure activity relationships. ACSS2 activity was measured in cell free and cellular assays by monitoring the conversion of radiolabelled acetate to biomass in depleted oxygen environments and in combination with chemotherapeutics such as doxorubicin. RESULTS Our medicinal chemistry campaign yielded several highly potent ACSS2 specific enzyme inhibitors with good pharmacokinetic properties in the rodent. Our lead compound CRD1400 is an ACSS2 specific inhibitor with low nanomolar potency and kills acetate dependent tumor cells and sensitizes several tumor cell lines to the chemotherapeutic doxorubicin. CRD1400 is has been selected for further assessment in patient derived xenograft models based on its potency for increased cell killing under hypoxic conditions. CONCLUSIONS Inhibition of the enzyme ACSS2 depletes tumor cells of the nutrient acetate and provides a new modality of attacking metabolic vulnerabilities in tumors. When used in combination, CRD1400 reduces chemotherapeutic resistance that arises in low oxygen environments common in tumors and enhances the potency of commonly used cancer medication. Citation Format: Monali Banerjee, Sourav Basu, Debjani Chakraborty, Ritesh Shrivastava, Sandip Middya, Rajib Ghosh, Dharmendra Yadav, Arghyotri Sinha, Avratanu Das, Navin Pandit, Mohinder Naiya, Sabyasachi Debnath, Sangeeta Dey, Debottam Dasgupta, Nirmal Das, Anuj Singh, Dipayan Sarkar, Sukanya Sarkar, Ganesh Narasipuram, Nagaswami Mane, Nicky Singhal, Nidhi Rawat, Anindita Middya, Arjun Surya. Small molecule ACSS2 inhibitors target acetate metabolizing tumor cells in hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3510.

Association of upregulation of serine and one carbon metabolism genes with shorter recurrence-free and overall survival in urothelial bladder cancer (UBC).

477 Background: Antimetabolites (e.g. methotrexate and gemcitabine) are not frequently used in the treatment of most solid tumors, but are effective in the treatment of UBC. Rapid cancer cell proliferation relies on an abundance of serine-derived one carbon units to support macromolecule synthesis. Specifically, PHGDH, which encodes a key enzyme of de novo serine synthesis, is amplified in breast cancer and in melanoma, and small molecule inhibitors of enzymes in this pathway are in early clinical development. However, the enzymes of serine and one carbon metabolism have not been widely investigated in UBC. Methods: We conducted an observational analysis of The Cancer Genome Atlas UBC cohort, focusing on gene expression data from a targeted panel indicated by Yang, et al. to be involved in serine and one carbon metabolism. Univariate Cox proportional hazard models were utilized to identify genes impacting OS and RFS, and a subsequent multivariate model was employed to control for inter-gene associations. Results: Expression data from 14 genes were analyzed among 436 UBC patients, of whom complete data were available for 422. At a median follow-up of 17 months, 188 of 422 patients had died. On univariate analysis, 7 of 14 genes were significantly associated with OS: PHGDH, PSPH, MTHFD1, MTHFD2, MTHFD1L, MTHFD2L, and ALDH1L2 (all P < 0.05). Interestingly, overexpression was associated with worse OS for all but one gene, MTHFD2L (HR 0.74), which is known to be underexpressed by cancer cells in favor of MTHFD2 (HR 1.21). In multivariate analysis, overexpression of PHGDH (HR 1.19, P = 0.008), MTHFD1 (HR 1.33, P = 0.041), and ALDH1L2 (HR 1.21, P < 0.001) were independent predictors of poor survival. RFS analysis was limited by missing data; nevertheless, univariate analyses found MTHFD1, MTHFD2, MTHFD1L, MTHFD2L, and ALDH1L2 to be associated with RFS (all P < 0.05). Conclusions: Within the limits of this observational study, these data suggest that serine and one carbon metabolism is important in the progression and prognosis of muscle-invasive bladder cancer. Subsequent in vitro analyses are needed to validate the prognostic and therapeutic significance of these findings.

Mechanistic enzymology in drug discovery: a fresh perspective.

Given the therapeutic and commercial success of small-molecule enzyme inhibitors, as exemplified by kinase inhibitors in oncology, a major focus of current drug-discovery and development efforts is on enzyme targets. Understanding the course of an enzyme-catalysed reaction can help to conceptualize different types of inhibitor and to inform the design of screens to identify desired mechanisms. Exploiting this information allows the thorough evaluation of diverse compounds, providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. This review highlights the rationale for conducting high-quality mechanistic enzymology studies and considers the added value in combining such studies with orthogonal biophysical methods.