Modern Medicinal Chemistry

Molecular recognition studies with biological receptors: structure-based de novo design of nonpeptidic enzyme inhibitors

Last updated: August 25, 2017

Selected References

This modern medicinal chemistry program aims at establishing rational lead generation as an intelligent, economic alternative to combinatorial and high-throughput screening approaches. It focuses at expanding the understanding of biological molecular recognition, generating detailed information on the strength of individual bonding interactions and their contribution to the overall free energy of complexation. Such knowledge is widely applicable to other medicinal chemistry projects.

The strong performance of our rational, structure-based design approach is illustrated by the following: in the first eight projects brought to the stage of biological testing, the first designed compounds showed an inhibitory activity (Ki or IC50) between 1 and 16 micromolar. Within 1-2 X-ray structure-based lead optimization cycles, single-digit nanomolar activities have been reached in all advanced projects. Whereas rational structure-based inhibitor design and synthesis are done in our laboratories, biological testing and protein crystallography are pursued in collaborations.

Actual targets in this research are, among others:

• t-RNA guanine transglycosylase (TGT), a target in antibacterial therapy against shigellosis (in collaboration with Prof. G. Klebe, Univ. Marburg). Shown is the X-ray crystal structure of the potent inhibitor 3 (inhibitory constant Ki = 6 nM) within the active site of TGT. The central lin-benzoguanine scaffold is sandwiched between a methionine and a tyrosine (not shown). The naphthyl side chain points into the pocket which hosts ribose33 of bound tRNA. Nucleic acid binding is efficiently inhibited by this ligand. There is strong evidence that the 2-aminoimidazole moiety is protonated and that complexation is enhanced by charge-assisted H-bonding. The achievement of single-digit nanomolar inhibition constants is a milestone towards the development of new, specific drugs against Shigellosis, a neglected disease of the third world.

a) Crystal structure of naphthyl-substituted 2-amino-lin-benzoguanine ligand bound to TGT, resolution 1.95 Å. b) Superimposition of the ligand and the natural substrate preQ1 bound to the active site of TGT and engaging in charge-assisted hydrogen bonding with E235, mediated by the peptide bond L231-A232

Some of our aims in the TGT project, that are all guided by structure-based design, are:

A first successful approach towards replacing the mentioned water cluster involves an ammonium ion linker as shown by X-ray crystallography for the cyclohexane-bearing ligand with an activity of Ki = 4 nM. Recently, we found that furanoside sugars are also suitable for replacing the water which solvates the two catalytic aspartates D102 and D280 near the polar ribose-34 pocket of the enzyme.  X-ray co-crystal structures of the TGT-furanoside ligand complexes revealed that the sugars occupy the phosphate binding site, and we have started a program to explore whether neutral small sugar derivatives might be general phosphate surrogates.

The ligand occupies, with the lin-benzoguanine core, the nucleobase pocket and an ammonium linker crosses the ribose 34 pocket to position the cyclohexyl ring onto a hydrophobic patch shaped by the side chains of Val282, Val45, and Leu68.

New Targets against Malaria. Malaria, a life-threatening disease caused by parasites of the genus Plasmodium, kills each year more than one million people, and more than 500 million clinical cases are registered annually. The recent emergence of multi-drug resistent strains of Plasmodium falciparum, the parasite that causes the deadliest form of malaria, demands the urgent development of new therapeutic agents with novel modes of action.

A variety of new targets is available in the search for innovative antimalarial therapies. Specific objectives in our research include preventing Hemoglobin degradation by P. Falciparum: Inhibition of the Plasmepsins and the Falcipains. During its stay in the human body, the parasites infect erythrocytes and catabolize hemoglobin to provide nutrient for its own growth and maturation. Hemoglobin degradation is initiated by a series of aspartic proteases, the plasmepsins (PMs), followed by degradation to smaller fragments by cysteine proteases, the falcipains (FPs). Because degradation of hemoglobin is vital for parasite survival, inhibition of these two classes of proteases offers a valid approach for the development of new therapeutic agents in our laboratory. Biological study suggests that both the four PMs and the three FPs need to be inhibited to starve the parasite. We wish to validate this proposal and develop potent and selective inhibitors for each class of proteases. This worl is pursued in collaboration with Actelion, Basel.

The figure below shows potent inhibitors of the aspartic proteases Plasmepsin II and IV. Optimal binding is achieve if the alkyl chain entering the flap vector of the proteins features a volume occupancy of around 55%, thereby validating the 55% rule by Mecozzi and Rebek for a biological binding site. Also, the data suggest that longer alkyl chains (> C7) fold in order to fit into the flap pocket.

Left above: Overlay of PM II X-ray crystal structures in the pepsin-like flap-closed conformation (PDB code: 1LF3, yellow) and the flap-open conformation (PDB code: 2BJU, red). In the latter, the Trp41 side chain rotates by 120° to open the flap pocket. Right: Illustration of the proposed binding mode.The n-heptyl chain is the longest that fits into the open flap pocket without contortion, in the all-anti conformation.


Left: IC50 values (nM) for PM II (squares) and PM IV (triangles) as a function of the number of C-atoms n in the n-alkyl chain flap vector. Right: Inhibition of PM II and PM IV and packing coefficients PCs calculated for PM II. Best binding is clearly observed at a volume occupancy of around 55%.

Preventing Isoprenoid Biosynthesis by the Non-Mevalonate Pathway in P. Falciparum: Inhibition of IspE and IspF. The non-mevalonate pathway for the biosynthesis of isoprenoids was discovered in the early 1990s and uses seven enzymes to transform pyruvate and glyceraldehyde 3-phosphate into the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). It is used exclusively by the Plasmodium parasites. Mammals, on the other hand, only use the alternative mevalonate pathway. Hence, the development of small-molecule inhibitors for enzymes of the non-mevalonate pathway constitutes a promising approach to selectively hit the malarial parasite. The pathway has previously been fully validated as antimalarial target, by inhibition of the second enzyme (IspC) with a small phosphonate, fosmidomycin. We propose the structure-based design of inhibitors of the fourth and fifth enzymes in the pathway, IspE (4-diphosphocytidyl-2C-methyl-D-erythritol kinase, EC and IspF (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, EC In preliminary work, we prepared drug-like inhibitors of IspE and IspF which are the first non-phosphonate, non-phosphate inhibitors of any of the enzymes in the pathway. To achieve high selectivity against human kinases, the inhibitors of the kinase IspE occupy the substrate and not the ATP site. These first-generation ligands will be systematically optimized and a new bi-substrate inhibition approach for IspE tested. This work is pursued in collaboration with Prof. A. Bacher (TU Munich) and Prof. W. N. Hunter (Univ. Dundee).


Top left: Active site of E. coli IspE in the ternary complex with CDP-ME and ATP analog AppNp (PDB code: 1OJ4). Bottom left: Design of the first-generation inhibitors for IspE. Top right: Proposed binding of the cyclopropyl moiety into the hydrophobic sub-pocket at the cytidine site of E. coli IspE. Bottom right: Activities of the new ligands with an SAR in clear agreement with the proposed binding of residue R in the small sub-pocket.

New Targets against African Sleeping Sickness

In one aspect of our work, we target the development of efficient ligands for Trypanothione Reductase (TR), an essential enzyme of trypanosomatids and therefore a promising target for the development of new drugs against African sleeping sickness and Chagas' disease. We prepared a series of diaryl sulfide-based ligands and, using computer modeling, we revised the binding model for this class of TR inhibitors. In vitro studies (collaboration with Prof. Reto Brun at the Swiss Tropical Institute (SPHTI) and with Prof. Luise Krauth-Siegel at the University of Heidelberg) showed IC50 values in the low micromolar to submicromolar range against Trypanosoma brucei rhodesiense and, unexpectedly, also against the malaria parasite Plasmodium falciparum.

Proposed binding mode of a new potent inhibitor against Trypanothione Reductase (TR).

In a second approach, we pursue the selective inhibition of an essential cysteine protease of Trypanosoma brucei, rhodesain, a cathepsin L-like hydrolase of T. brucei, which exhibits a crucial role in the parasite life-cycle.

Based on available X-ray crystal structures, we utilized computer-aided 3D modeling to design small drug-like molecules to occupy the various apolar pockets of the active sites. The general structure of cysteine protease inhibitors contains prevalently an electrophilic moiety to form a reversible, covalent thioimidate intermediate with the catalytic cysteine. We therefore opted specifically for inhibitors featuring a nitrile residue as electrophilic head group.

Based on our structure-based design approach, we prepared a series of functionalized triazine nitrile inhibitors which were tested (in collaboration with Prof. T. Schirmeister, Univ. Würzburg) against falcipain-2 and rhodesain in standard fluorescence-based assays.  Promising inhibitory activities down to Ki = 2 nm for rhodesain were obtained for this first generation triazine nitrile-based inhibitors.  Biological assays showed in most cases good selectivity against closely related human enzymes, and in vitro studies at the Swiss Public Health and Tropical Institute (SPHTI) in collaboration with the group of Prof. R. Brun revealed moderate activity of the synthesized compounds against the parasites. These results are now followed by detailed optimization studies in order to diminish off-target effects and improve bioavailability and ADMET parameters.  It is noticeable that some of these ligands are also strong inhibitors of Falcipain-2, a hemoglobinase of P. falciparum.

The ligand is bound by rhodesain from T. Brucei with a Ki-value of 2 nM. It is covalently bound as thioimidate to the catalytic cysteine of the enzyme and fills the S2 and S3 pockets.

Molecular recognition studies with the cysteine proteases human cathepsin L (hCatL) and rhodesain.

In collaboration with scientists from Roche, Basel, we found that the introduction of fluorine substituents on benzene rings of triazine nitrile inhibitors enhances the π-stacking of these rings with peptide amide bonds at the entrance of the S3 pocket up to 13-fold.  This "fluorine scan" shows that degree and pattern of fluorine substitution matter, which is explained in experimental/computational study by changes in lipophilicity, dipolar and quadrupolar interactions, and additional protein-ligand interactions beyond the arene-amide stacking.

We also conducted a "heteroarene scan" by introducing heteroarene substituents in the S3 pocket of hCatL and rhodesain.  Heteroarenes strongly enhance the binding affinity of triazine nitrile inhibitors over their corresponding aromatic hydrocarbon analogues.  The heterocycles undergo π-stacking on the peptide bonds located at the entrance of the pocket.  Inhibitory activities vary over three orders of magnitude in a series of ligands bearing bicyclic heteroarenes as S3 pocket vectors.  The high binding affinity of sulfur-containing heterocycles was explained by intramolecular S...O chalcogen bonding in a detailed molecular recognition analysis which included co-crystal structures, interaction network analysis, and conformational analyses.

Predicted binding mode of the benzothiophene ligand in hCatL.  The protein coordinates are taken from the co-crystal structure of the corresponding imidazopyridine ligand (PDB-ID: SMAJ, 1.0 Å resolution).

Free Energy calculations for the prioritization of hCatL inhibitors. In a collaborative effort involving scientists at Roche Basel, Schrödinger New York, the Univ. of Mainz, and ETH, free energy perturbation (FEP) calculations were conducted to predict the activity of 36 new ligands of hCatL, in which the S2 pocket vector was systematically varied.  In this work, the FEP calculations preceeded the experimental work after validation for the enzyme-ligand binding system.  The study showed that FEP can be a useful approach to guide medicinal chemistry optimization.

We reported a new family of enantiopure inhibitors for protein kinase A (PKA). They complex to the ATP site, with inhibitory activities (Ki down to the low single-digit nanomolar range. Besides binding to the hinge and the ribose pocket, the ligands also interact in a multi-dimensional interaction network with the glycine-rich loop, which has been neglected in kinase inhibitor design. This network of orthogonal dipolar interactions, dispersive contacts, amide-π stacking, together with water replacement makes an important contribution to the overall ligand binding affinity. This is the first example, how to efficiently harvest binding energy through interactions of ligands with the glycin-rich loop in protein kinases. Upon adjustment to other kinase targets, this strategy should be a broadly applicable.

Co-crystal structure of an enantiopure ligand with protein kinase A (PKA) interacting with a 2-bromothienyl ring with the glycine-rich loop. Ki = 0.9 nM.

Several enzymes of the folate cycle are well-known and clinically validated antimalarial targets. To our surprise, efficient inhibitors had not been previously described for one of the key enzymes of the cycle, serine hydroxymethyltransferase (SHMT). We prepared a series of pyrazolopyran ligands as highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT, with cell-based activities (EC50 values) in the low single-digit nanomolar range against blood-state Pf and liver-stage P. berghei (Pb) cells and high selectivity against mammalian cell lines. These ligands are now further optimized using structure-based design, as co-crystal structures with PvSHMT were solved.