Simona M. Ceccarelli

A Real-World Perspective on Molecular Design.

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

Carnitine palmitoyltransferase (CPT) modulators: a medicinal chemistry perspective on 35 years of research.

The metabolism of fatty acids, including their absorption, storage, mobilization, synthesis, and catabolism, has been the origin and target of innumerable drugs and pharmacological tools and the focus of countless research programs. The oxidation of fatty acids (FAO) is one of the most important cellular energy sources, and a pharmacological control on this process could have interest for a variety of therapeutic applications. In diabetes, the reduced sensitivity to insulin causes excessive release of fatty acids from the adipose tissue and increases their oxidation rate. This compounds the defects in tissue glucose uptake by promoting underutilization of glucose in the cells and overproduction of glucose by the liver, particularly due to excessive gluconeogenesis. In the failing, overloaded heart, generation of energy by FAO poses heavy demands on the diminishing oxygen supply and promotes accumulation of potential toxic metabolites. FAO inhibition in cancer cells that are heavily dependent on lipids as energy source has the potential to shut down their growth. In dislipidemic and/or obese patients, on the other hand, increasing the FAO rate could curb the levels of circulating lipids and reduce tissue lipid storage, impacting favorably on body weight and the development of insulin resistance. Finally, elements of the FAO machinery in the brain contribute to central control of energy homeostasis and feeding behavior. Most of the oxidation of long chain fatty acids (LCFAs) to acetyl-CoA occurs in the mitochondrial matrix. LCFAs are first converted to their CoA esters by the ATP dependent acyl-CoA synthases in the outer mitochondrial membrane, the cytosol, and the endoplasmic reticulum; however, themitochondrialmembrane is not permeable to long chain acyl-CoA (over ∼C12). The mechanism by which LCFA access the mitochondrial matrix was elucidated by the pioneering studies of Fritz andYue andMcGarry and Foster and is illustrated in Figure 1. Acyl-CoAs are converted to acylcarnitine derivatives by the enzyme carnitine palmitoyltransferase 1 (CPT1) on the cytosolic face of the external mitochondrial membrane. Acylcarnitines are substrates for the shuttle-transporter carnitine acylcarnitine translocase (CACT), which mediates the transit of acylcarnitines from the cytosol to the matrix and the transport of free carnitine in the opposite direction. Once inside the mitochondrial membranes, acylcarnitines are reconverted to acyl-CoA by the enzyme carnitine palmitoyltransferase 2 (CPT2) and can thus enter the FAO cycle. McGarry and Foster established in 1980, on the basis of careful experimental data, that CPT1 is the controlling element of FAO rate and ketogenesis. The term “rate-controlling” is more appropriate than “rate-limiting”, which has been used by some authors. Indeed, CPT1 activity is regulated by a considerable number of signals, which deliver feedback on the energy requirements of the organism and can vary over an extremely wide range. The variable basal activity of CPT1 and the difficulties in obtaining the isolated enzyme in active form have made it difficult to dissect the contribution of the various elements of the CPT system to FA transport and oxidation rate. FAO can also occur in microsomes and peroxisomes, which process LCFAdown to theC8 length, after which they can enter the mitochondria via passive transport. Microsomes and peroxisomes possess their own CPTs, enzymes that are not well characterized but possibly are very similar if not identical to the mitochondrial CPT enzymes. Peroxisomal oxidation can be induced in situations where themitochondrial oxidation does not have sufficient capacity (for example, by high fat diet), and it has been demonstrated that the peroxisomal CPT is subject to similar controlling elements as the mitochondrial CPT system. The contribution of peroxisomal CPT activity to observed FAO rates in cellular systems upon CPT inhibition is an incognita. The lack of molecular modulators with well-defined activity and selectivity has been an added hurdle. Since the late 1970s, a few small molecules affecting the CPT enzymes, particularly inhibitors, have been identified. The oxirane carboxylic acids described by Tutwiler, Wolf, Sherratt, and Eistatter (BGLCF GmbH and McNeil) and the aminocarnitine derivatives described by Giannessi (Sigma Tau), Gandour, Anderson, and Griffith have been the most prominent and well characterized examples. These compounds have been used in an impressive number of in vitro and in vivo assays (including human clinical studies), assessing in particular potential effects in diabetes and cardiac failure. Nevertheless, or perhaps for this reason, a quantitative, consistent picture of relative and absolute activity, in vivo potency, selectivity, safety, and therapeutic potential of the various molecular entities is difficult to draft. In view of the complexity of the system, its sensitivity to a great number of factors, and the number of different setups that have been used to investigate its behavior, this is perhaps not surprising. It is important to stress at this stage that all compounds for which data are reported in the literature are nonselective inhibitors and affect more than one isoform of CPT (besides known or unknown off-target effects). Interest in this target, as documented by scientific literature, has been slowly but steadily growing in the past decades (Figure 2). A few reviews have been compiled that cover CPT inhibitors. This review will attempt to appraise the wide and tangled field of CPT modulators from the perspective of medicinal chemistry, using the chemotypes of CPT-interacting agents that have been described in the literature as a guiding beacon. Given the difficulty in obtaining quantitative comparable

Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators: Discovery of 2-Chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (Basimglurant, RO4917523), a Promising Novel Medicine for Psychiatric Diseases

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinsons disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.

LC–SPE–NMR–MS: a total analysis system for bioanalysis

Liquid chromatography (LC)-solid-phase extraction (SPE)-nuclear magnetic resonance (NMR)-mass spectrometry (MS) coupling is a key technology for fast and thorough structure elucidation of valuable mass-limited samples. Laborious serial isolation and purification procedures of metabolites, byproducts or impurities from complex biomatrices, natural product extracts or other mixtures of several components can be circumvented by the use of this integrated modular system. This combination of high-end analytical technology significantly accelerates the structure-elucidation process for valuable samples present in minute quantities in mixtures. The information depth is significantly increased by the concurrent availability of NMR and MS data of one chromatographic peak. Thus, this flexible technique is well on its way to becoming the gold standard in analytical chemistry of mixtures. LC-SPE-NMR-MS overcomes the limitations of directly coupled LC-NMR. Full flexibility regarding chromatographic conditions and NMR acquisition is gained by this modular technique. LC-SPE-NMR-MS allows for a rapid structure-elucidation process that would not be possible on the basis of MS or NMR data alone.

Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein.

Carnitine palmitoyl transferase 2 (CPT‐2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y‐shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT‐2 is a monotopic membrane protein and was solubilized by β‐octylglucoside (β‐OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β‐OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT‐2 (rCPT‐2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2–20 μM. New X‐ray structures and docking models of rCPT‐2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy‐driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X‐ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

Metabolite Identification via LC-SPE-NMR-MS of the In vitro Biooxidation Products of a Lead mGlu5 Allosteric Antagonist and Impact on the Improvement of Metabolic Stability in the Series

Detailed information on the metabolic fate of lead compounds can be a powerful tool for an informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The combination of high performance liquid chromatography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of on‐line solid‐phase extraction (SPE) into a integrated LC‐SPE‐NMR‐MS system have improved enormously the detection limits of this approach. The new technology platform allows the analysis of complex mixtures from microsome incubations, combining low material requirements with relatively high throughput. Such characteristics make it possible to thoroughly characterize metabolites of selected compounds at earlier phases along the path to lead identification and clinical candidate selection, thus providing outstanding guidance in the process of eliminating undesired metabolism and detecting active or potentially toxic metabolites. Such an approach was applied at the lead identification stage of a backup program on metabotropic glutamate receptor 5 (mGlu5) allosteric inhibition. The major metabolites of a lead 5‐aminothiazole‐4‐carboxylic acid amide 1 were synthesized and screened, revealing significant in vitro activity and possible involvement in the overall pharmacodynamic behavior of 1. The information collected on the metabolism of the highly active compound 1 was pivotal to the synthesis of related compounds with improved microsomal stability.

Design and synthesis of selective, dual fatty acid binding protein 4 and 5 inhibitors.

Dual inhibition of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is expected to provide beneficial effects on a number of metabolic parameters such as insulin sensitivity and blood glucose levels and should protect against atherosclerosis. Starting from a FABP4 selective focused screening hit, biostructure information was used to modulate the selectivity profile in the desired way and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. With very good pharmacokinetic properties and no major safety alerts, compound 12 was identified as a suitable tool compound for further in vivo investigations.