P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.     

Novel non-active site inhibitor of Cryptosporidium hominis TS-DHFR identified by a virtual screen

The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.     

Design of novel, potent, and selective human beta-tryptase inhibitors based on alpha-keto-[1,2,4]-oxadiazoles

A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.     

Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors

Synthesis and SAR are described for a structurally distinct class of DPP–IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative (S,S,S)-2g possesses the best fit in the S1 pocket. However, (S,S,S)-2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.     

2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hydrophilicity and selectivity

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent exposed area of the enzyme provides an option to install polar groups (R¹) the 5-position of 2-aminoquinazolin-4(3H)-one to inhibitors such as carboxylic acid without scarifying enzymatic potency. Moreover, introduction of R¹ substituents increased selectivity factors of compounds in this series up to 100-fold for Plm II, IV vs CatD inhibition. The introduction of flap pocket substituent (R²) at 7-postion of 2-aminoquinazolin-4(3H)-one allows to remove Ph group from THF ring without notably impairing Plm inhibitory potency. Based on these findings, inhibitors were developed, which show Plm II and IV inhibitory potency in low nanomolar range and remarkable selectivity against Cathepsin D along with decreased lipophilicity and increased solubility.     

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC₅₀ = 7.7–11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.     

Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors: Part 1

A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crystal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submicromolar cellular potency.     

Design and synthesis of thiazolylhydrazone derivatives as inhibitors of chitinolytic N-acetyl-β-d-hexosaminidase

N-acetyl-β-d-hexosaminidase (Hex) is potential target for pesticide design. Here, a series of thiazolylhydrazone derivatives were designed, synthesized and evaluated as competitive inhibitors of OfHex1, a Hex from the agricultural pest Ostrinia furnacalis. The derivative 3k, with a (benzyloxy)methyl group at the N3 atom, demonstrated greater potency with a K_i of 10.2?µM. Molecular docking analysis indicated that the (benzyloxy)methyl group of 3k was bound to a previously unexplored pocket formed by Loop478-496. Then further optimization around naphthalene ring led to find the more potency substituent phenyl. The derivative 7, with phenoxyethyl group at R₁ and a phenyl group at R₂, demonstrated an augmented potency with a K_i of 2.1?µM. Molecular docking analysis indicated that 7 was bound to the active pocket of OfHex1 more favorably than 3k. This work suggests a novel scaffold for developing specific Hex inhibitors.     

High affinity sialoside ligands of myelin associated glycoprotein

Myelin associated glycoprotein (Siglec-4) is a myelin adhesion receptor, that is, well established for its role as an inhibitor of axonal outgrowth in nerve injury, mediated by binding to sialic acid containing ligands on the axonal membrane. Because disruption of myelin-ligand interactions promotes axon outgrowth, we have sought to develop potent ligand based inhibitors using natural ligands as scaffolds. Although natural ligands of MAG are glycolipids terminating in the sequence NeuAcα2-3Galβ1-3(±NeuAcα2−6)GalNAcβ-R, we previously established that synthetic O-linked glycoprotein glycans with the same sequence α-linked to Thr exhibited ∼1000-fold increased affinity (∼1 μM). Attempts to increase potency by introducing a benzoylamide substituent at C-9 of the α2-3 sialic acid afforded only a two-fold increase, instead of increases of >100-fold observed for other sialoside ligands of MAG. Surprisingly, however, introduction of a 9-N-fluoro-benzoyl substituent on the α2-6 sialic acid increased affinity 80-fold, resulting in a potent inhibitor with a K_d of 15 nM. Docking this ligand to a model of MAG based on known crystal structures of other siglecs suggests that the Thr positions the glycan such that aryl substitution of the α2-3 sialic acid produces a steric clash with the GalNAc, while attaching an aryl substituent to the other sialic acid positions the substituent near a hydrophobic pocket that accounts to the increase in affinity.     

Action at a Distance: MUTATIONS OF PERIPHERAL RESIDUES TRANSFORM RAPID REVERSIBLE INHIBITORS TO SLOW,TIGHT BINDERS OF CYCLOOXYGENASE-2*

Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin G₂. The inhibitory activity of rapid, reversible COX inhibitors (ibuprofen, naproxen, mefenamic acid, and lumiracoxib) demonstrated a significant increase in potency and time dependence of inhibition against double tryptophan murine COX-2 mutants at the 89/90 and 89/119 positions. In contrast, the slow, time-dependent COX inhibitors (diclofenac, indomethacin, and flurbiprofen) were unaffected by those mutations. Further mutagenesis studies suggested that mutation at position 89 was principally responsible for the changes in inhibitory potency of rapid, reversible inhibitors, whereas mutation at position 90 may exert some effect on the potency of COX-2-selective diarylheterocycle inhibitors; no effect was observed with mutation at position 119. Several crystal structures with or without NSAIDs indicated that placement of a bulky residue at position 89 caused a closure of a gap at the lobby, and alteration of histidine to tryptophan at position 90 changed the electrostatic profile of the side pocket of COX-2. Thus, these two residues, especially Val-89 at the lobby region, are crucial for the entrance and exit of some NSAIDs from the COX active site.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Think Twice: Understanding the High Potency of Bis(phenyl)methane Inhibitors of Thrombin

Successful design of potent and selective protein inhibitors, in terms of structure-based drug design, strongly relies on the correct understanding of the molecular features determining the ligand binding to the target protein. We present a case study of serine protease inhibitors with a bis(phenyl)methane moiety binding into the S3 pocket. These inhibitors bind with remarkable potency to the active site of thrombin, the blood coagulation factor IIa. A combination of X-ray crystallography and isothermal titration calorimetry provides conclusive insights into the driving forces responsible for the surprisingly high potency of these inhibitors. Analysis of six well-resolved crystal structures (resolution 1.58-2.25 Å) along with the thermodynamic data allows an explanation of the tight binding of the bis(phenyl)methane inhibitors. Interestingly, the two phenyl rings contribute to binding affinity for very different reasons — a fact that can only be elucidated by a structure-based approach. The first phenyl moiety occupies the hydrophobic S3 pocket, resulting in a mainly entropic advantage of binding. This observation is based on the displacement of structural water molecules from the S3 pocket that are observed in complexes with inhibitors that do not bind in the S3 pocket. The same classic hydrophobic effect cannot explain the enhanced binding affinity resulting from the attachment of the second, more solvent-exposed phenyl ring. For the bis(phenyl)methane inhibitors, an observed adaptive rotation of a glutamate residue adjacent to the S3 binding pocket attracted our attention. The rotation of this glutamate into salt-bridging distance with a lysine moiety correlates with an enhanced enthalpic contribution to binding for these highly potent thrombin binders. This explanation for the magnitude of the attractive force is confirmed by data retrieved by a Relibase search of several thrombin-inhibitor complexes deposited in the Protein Data Bank exhibiting similar molecular features.Special attention was attributed to putative changes in the protonation states of the interaction partners. For this purpose, two analogous inhibitors differing mainly in their potential to change the protonation state of a hydrogen-bond donor functionality were compared. Buffer dependencies of the binding enthalpy associated with complex formation could be traced by isothermal titration calorimetry, which revealed, along with analysis of the crystal structures (resolution 1.60 and 1.75 Å), that a virtually compensating proton interchange between enzyme, inhibitor and buffer is responsible for the observed buffer-independent thermodynamic signatures.     

New pyrazolyl and thienyl aminohydantoins as potent BACE1 inhibitors: exploring the S2' region

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer's disease. SAR studies of the S2' region of the BACE1 ligand binding pocket with pyrazolyl and thienyl P2' side chains are reported. These analogs exhibit low nanomolar potency for BACE1, and demonstrate >50- to 100-fold selectivity for the structurally related aspartyl proteases BACE2 and cathepsin D. Small groups attached at the nitrogen of the P2' pyrazolyl moiety, together with the P3 pyrimidine nucleus projecting into the S3 region of the binding pocket, are critical components to ligand's potency and selectivity. P2' thiophene side chain analogs are highly potent BACE1 inhibitors with excellent selectivity against cathepsin D, but only modest selectivity against BACE2. The cell-based activity of these new analogs tracked well with their increased molecular binding with EC(50) values of 0.07-0.2 μM in the ELISA assay for the most potent analogs.     

Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.     

Structural Insights into Central Hypertension Regulation by Human Aminopeptidase A

Hypertension is regulated through both the central and systemic renin-angiotensin systems. In the central renin-angiotensin system, zinc-dependent aminopeptidase A (APA) up-regulates blood pressure by specifically cleaving the N-terminal aspartate, but not the adjacent arginine, from angiotensin II, a process facilitated by calcium. Here, we determined the crystal structures of human APA and its complexes with different ligands and identified a calcium-binding site in the S1 pocket of APA. Without calcium, the S1 pocket can bind both acidic and basic residues through formation of salt bridges with the charged side chains. In the presence of calcium, the binding of acidic residues is enhanced as they ligate the cation, whereas the binding of basic residues is no longer favorable due to charge repulsion. Of the peptidomimetic inhibitors of APA, amastatin has higher potency than bestatin by fitting better in the S1 pocket and interacting additionally with the S3′ subsite. These results explain the calcium-modulated substrate specificity of APA in central hypertension regulation and can guide the design and development of brain-targeting antihypertensive APA inhibitors.     

Crystal structures of novel non-peptidic, non-zinc chelating inhibitors bound to MMP-12

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and has been implicated in diseases such as emphysema and chronic obstructive pulmonary disease (COPD). It is therefore an attractive target for therapeutic agents.As part of a structure-based drug design programme to find new inhibitors of MMP-12, the crystal structures of the MMP-12 catalytic domain (residues 106-268) complexed to three different non-peptidic small molecule inhibitors have been determined. The structures reveal that all three ligands bind in the S1′ pocket but show varying degrees of interaction with the Zn atom. The structures of the complexes with inhibitors CP-271485 and PF-00356231 reveal that their central morpholinone and thiophene rings, respectively, sit over the Zn atom at a distance of approximately 5 Å, locating the inhibitors halfway down the S1′ pocket. In both of these structures, an acetohydroxamate anion, an artefact of the crystallisation solution, chelates the zinc atom. By contrast, the acetohydroxamate anion is displaced by the ligand in the structure of MMP-12 complexed to PD-0359601 (Bayer), a potent zinc chelating N-substituted biaryl butyric acid, used as a reference compound for crystallisation. Although a racemate was used for the crystallisation, the S enantiomer only is bound in the crystal. Important hydrophobic interactions between the inhibitors and residues from the S1′ pocket are observed in all of the structures. The relative selectivity displayed by these ligands for MMP-12 over other MMP family members is discussed.     

Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1′ pocket.     

Examination of halogen substituent effects on HIV-1 integrase inhibitors derived from 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-ones and 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones

Using 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one and 4,5-dihydroxy-1H-isoindole-1,3(2H)-dione based HIV-1 integrase inhibitors as display platforms, we undertook a thorough examination of the effects of modifying the halogen substituents on a key benzyl ring that is hypothesized to bind in a hydrophobic pocket of the integrase·DNA complex. Data from this study suggest that in general dihalo-substituted analogues have higher potency than monohalo-substituted compounds, but that further addition of halogens is not beneficial.     

Evaluation of dipeptide nitriles as inhibitors of rhodesain,a major cysteine protease of Trypanosoma brucei

A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8 μM) the compounds represent promising starting points for new rhodesain inhibitors.