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Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Low solubility of drugs represents a major challenge during research and development. Ways to overcome this are either focused on formulation development or optimization of the molecular structure of the drug. The latter is not only governed by the constitution of the molecule but also by its stereochemistry. Development of enantiomers in contrast to racemic mixtures has become the state of the art over the last decades as this leads to higher potency and selectivity. Thus, enantiopure drugs require lower doses compared to their racemates. Additionally, selecting one enantiomer also leads to improved solubility of the drug compared to its racemic compound. While this effect is well known for enantiomers and racemic compounds where chirality is introduced via a chiral central atom, here we describe the first case where improved solubility is realized by selecting an axially chiral atropisomer.

7,7′-Disubstituted 2,2′-methylenedioxy-1,1′-binaphthyls are highly efficient chirality inducers in nematic liquid crystals. The absolute configuration of these compounds is, however, hard to determine as they only crystallize as racemic mixtures. In this work a Vibrational Circular Dichroism (VCD) study is reported that provides an unambiguous determination of the absolute configuration of these compounds. An in-depth General Coupled Oscillator (GCO) analysis of the source of the VCD signal reveals that the unusual structure of these binaphthyl compounds inherently leads to strong and robust VCD bands. Combined with linear transit calculations, our VCD studies allow for the determination of key structural parameters.

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1μM) led to the most potent compound 27 (IC50: 0.038μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.

Proteasomes are large multi-subunit proteolytic complexes which are key components of the ubiquitin-dependent protein degradation pathway. The constitutive proteasome is an isoform which is expressed in most tissues, where it regulates protein homeostasis. In contrast, the immunoproteasome is predominantly expressed in hematopoietic cells and can be induced in non-hematopoietic cells by inflammatory stimuli such as IFNγ. The constitutive proteasome contains the catalytically different subunits β1c, β2c and β5c, which possess caspase-like, trypsin-like and chymotrypsin-like activity, respectively. These subunits are replaced in the immunoproteasome by β1i, β2i and β5i (LMP7, PSMB8) which display chymotrypsin-like, trypsin-like and chymotrypsin-like activity, respectively. The enhanced proteolytic capacity of the LMP7 subunit underpins the key function of the immunoproteasome in restoring homeostasis under conditions of elevated proteolytic or oxidative stress. Furthermore, the peptides generated by LMP7 are efficiently loaded onto MHC I and as such, the immunoproteasome plays an essential role in the adaptive immune response and inflammatory diseases. The clinically approved proteasome inhibitors Bortezomib, Ixazomib, or Carfilzomib indiscriminately inhibit multiple subunits of the constitutive proteasome and immunoproteasome, including LMP7, in a comparable potency range. The therapeutic applicability of these drugs is limited by the diverse toxicities that arise as a result of the inhibition of the constitutive proteasome in healthy tissues. To reduce toxicities associated with pan-proteasome inhibition, a drug discovery program was initiated to identify selective inhibitors of the LMP7 subunit of the immunoproteasome. A hit series containing the well-established α-aminoboronic acid scaffold, which interacts covalently and reversibly with the hydroxyl group of Thr-1 in the active site of LMP7, served as starting point. Co-crystallization of series representatives in complex with the 20S core particle of the human immunoproteasome guided rational optimization of both potency and selectivity. Based on the underlying structure activity relationship differences in the P1 and P3 pockets of the proteasome subunits were exploited to gain high specificity for LMP7. Further optimization of ADME and physicochemical properties led to the discovery of M3258, a potent, orally-bioavailable inhibitor of the LMP7 subunit demonstrating >500-fold selectivity against β1c, β2c, β5c, β1i and β2i. M3258 displayed favorable pharmacokinetics and demonstrated strong LMP7 inhibition and anti-tumor activity in several multiple myeloma xenografts, including models that were refractory to Bortezomib. Supported by favorable preclinical data, phase I assessment of M3258 is planned in 2019. Citation Format: Markus Klein, Michael Busch, Christina Esdar, Manja Friese-Hamim, Mireille Krier, Djordje Musil, Felix Rohdich, Michael Sanderson, Gina Walter, Oliver Schadt, Ugo Zanelli, Jianguo Ma. Discovery and profiling of M3258, a potent and selective LMP7 inhibitor demonstrating high efficacy in multiple myeloma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-054.

Large multifunctional peptidase 7 (LMP7, β5i, PSMB8) is a chymotrypsin-like proteolytic subunit of the immunoproteasome, which degrades ubiquitinated proteins and generates peptides for presentation on MHC class I. In contrast to the constitutive proteasome, which is broadly expressed, the immunoproteasome is specifically present in normal and malignant hematopoietic cells and can be induced in non-hematopoietic cells by inflammatory stimuli such as IFNγ. Pan-proteasome inhibitors like Bortezomib, which is approved in multiple myeloma and mantle cell lymphoma, indiscriminately inhibit the proteolytic activities of multiple subunits of the constitutive proteasome and immunoproteasome, including LMP7. Widespread proteasome inhibition is believed to underpin the adverse safety profiles of these agents and limit their therapeutic potential. Selective LMP7 targeting could achieve improved antitumor activity as a result of enhanced target inhibition, meanwhile circumventing the dose-limiting severe toxicities associated with pan-proteasome inhibitors. Based on the aforementioned hypothesis, a drug discovery program was initiated to identify selective inhibitors of LMP7. This led to the discovery of M3258, a covalent-reversible, potent, selective and orally-bioavailable inhibitor of LMP7. M3258 demonstrated strong in vivo antitumor activity, up to complete regression, in multiple myeloma xenograft models at daily oral doses as low as 1 mg/kg. This was associated with significant and prolonged suppression of tumor LMP7 activity. Furthermore, M3258 was efficacious in several multiple myeloma models that were refractory to Bortezomib. Subacute GLP toxicology studies with M3258, applied orally on a once-daily schedule, identified the lymphatic and hematopoietic systems in rat and dog and intestinal system in dog alone as main target organs. Importantly, M3258 was without effect on the peripheral and central nervous systems and cardiac and respiratory organs. Overall, M3258 demonstrated a superior preclinical therapeutic window and more restricted spectrum of toxicities compared to pan-proteasome inhibitors. Supported by robust preclinical data, clinical phase I assessment of M3258 in multiple myeloma patients is planned to begin in 2019. Citation Format: Michael Sanderson, Michael Busch, Christina Esdar, Manja Friese-Hamim, Mireille Krier, Jianguo Ma, Djordje Musil, Felix Rohdich, Willem Sloot, Gina Walter, Ugo Zanelli, Oliver Schadt, Markus Klein. First-time disclosure of M3258: A selective inhibitor of the immunoproteasome subunit LMP7 with potential for improved therapeutic utility in multiple myeloma compared to pan-proteasome inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr DDT02-01.

Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.

RAF kinase plays a critical role in the RAF-MEK-ERK signaling pathway and inhibitors of RAF could be of use for the treatment of various cancer types. We have designed potent RAF-1 inhibitors bearing novel bicyclic heterocycles as key structural elements for the interaction with the hinge region. In both series exploration of the SAR was focussed on the substitution of the phenyl ring, which binds to the induced fit pocket. Overall, it was confirmed that incorporation of lipophilic substituents was needed for potent Raf inhibition and a number of potent analogues were obtained.

Combinatorial chemistry and library design have been reconciled by applying simple medicinal chemistry concepts to virtual library design. The herein reported "Scaffold-Linker-Functional Group" (SLF) approach has the aim to maximize diversity while minimizing the size of a scaffold-focused library with the aid of simple molecular variations in order to identify critical pharmacophoric elements. Straightforward rules define the way of assembling three building blocks: a conserved scaffold, a variable linker, and a variable functional group. By carefully selecting a limited number of functional groups not overlapping in pharmacophoric space, the size of the library is kept to a few hundred. As an application of the SLF approach, a small-sized combinatorial library (320 compounds) was derived from the scaffold of the known phosphodiesterase 4 inhibitor zardaverine. The most interesting analogues were further prioritized for synthesis and enzyme inhibition by FlexX docking to the X-ray structure of the enzyme, leading to a 900-fold increased affinity within nine synthesized compounds and a single screening round.