DLiP-PPI library: An integrated chemical database of small-to-medium-sized molecules targeting protein-protein interactions.

Protein-protein interactions (PPIs) are recognized as important targets in drug discovery. The characteristics of molecules that inhibit PPIs differ from those of small-molecule compounds. We developed a novel chemical library database system (DLiP) to design PPI inhibitors. A total of 32,647 PPI-related compounds are registered in the DLiP. It contains 15,214 newly synthesized compounds, with molecular weight ranging from 450 to 650, and 17,433 active and inactive compounds registered by extracting and integrating known compound data related to 105 PPI targets from public databases and published literature. Our analysis revealed that the compounds in this database contain unique chemical structures and have physicochemical properties suitable for binding to the protein-protein interface. In addition, advanced functions have been integrated with the web interface, which allows users to search for potential PPI inhibitor compounds based on types of protein-protein interfaces, filter results by drug-likeness indicators important for PPI targeting such as rule-of-4, and display known active and inactive compounds for each PPI target. The DLiP aids the search for new candidate molecules for PPI drug discovery and is available online (https://skb-insilico.com/dlip).

Isostearyl Mixed Anhydrides for the Preparation of N-Methylated Peptides Using C-Terminally Unprotected N-Methylamino Acids.

Sustainable and efficient manufacturing methods for N-methylated peptides remain underexplored despite growing interest in therapeutic N-methylated peptides within the pharmaceutical industry. A methodology for the coupling of C-terminally unprotected N-methylamino acids mediated by an isostearic acid halide (ISTAX) and silylating reagent has been developed. This approach allows for the coupling of a wide variety of amino acids and peptides in high yields under mild conditions without the need for a C-terminal deprotection step in the process of C-terminal elongation. These advantages make this a useful synthetic method for the production of peptide therapeutics and diagnostics containing N-methylamino acids.

Structural States of Hdm2 and HdmX: X-ray Elucidation of Adaptations and Binding Interactions for Different Chemical Compound Classes.

Hdm2 (human MDM2, human double minute 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20 Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors.

In vitro and in vivo characterization of a novel, highly potent p53-MDM2 inhibitor.

Small molecule inhibitors of the p53-MDM2 protein complex are under intense investigation in clinical trials as anti-cancer agents, including our first generation inhibitor NVP-CGM097. We recently described the rational design of a novel pyrazolopyrrolidinone core as a new lead structure and now we report on the synthesis and optimization of this to provide a highly potent lead compound. This new compound displayed excellent oral efficacy in our preclinical mechanistic in vivo model and marked a significant milestone towards the identification of our second generation clinical candidate NVP-HDM201.

Discovery of a novel class of highly potent inhibitors of the p53-MDM2 interaction by structure-based design starting from a conformational argument.

The p53-MDM2 interaction is an anticancer drug target under investigation in the clinic. Our compound NVP-CGM097 is one of the small molecule inhibitors of this protein-protein interaction currently evaluated in cancer patients. As part of our effort to identify new classes of p53-MDM2 inhibitors that could lead to additional clinical candidates, we report here the design of highly potent inhibitors having a pyrazolopyrrolidinone core structure. The conception of these new inhibitors originated in a consideration on the MDM2 bound conformation of the dihydroisoquinolinone class of inhibitors to which NVP-CGM097 belongs. This work forms the foundation of the discovery of HDM201, a second generation p53-MDM2 inhibitor that recently entered phase I clinical trial.

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode.

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.

Discovery of a Dihydroisoquinolinone Derivative (NVP-CGM097): A Highly Potent and Selective MDM2 Inhibitor Undergoing Phase 1 Clinical Trials in p53wt Tumors.

As a result of our efforts to discover novel p53:MDM2 protein-protein interaction inhibitors useful for treating cancer, the potent and selective MDM2 inhibitor NVP-CGM097 (1) with an excellent in vivo profile was selected as a clinical candidate and is currently in phase 1 clinical development. This article provides an overview of the discovery of this new clinical p53:MDM2 inhibitor. The following aspects are addressed: mechanism of action, scientific rationale, binding mode, medicinal chemistry, pharmacokinetic and pharmacodynamic properties, and in vivo pharmacology/toxicology in preclinical species.

A distinct p53 target gene set predicts for response to the selective p53-HDM2 inhibitor NVP-CGM097.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.

Tetra-substituted imidazoles as a new class of inhibitors of the p53-MDM2 interaction.

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53-MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein-protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.

The central valine concept provides an entry in a new class of non peptide inhibitors of the p53-MDM2 interaction.

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein-protein interaction, we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53-MDM2 interaction.

New pyrazolo[1,5a]pyrimidines as orally active inhibitors of Lck.

A novel series of pyrazolo[1,5a]pyrimidines was optimized to target lymphocyte-specific kinase (Lck). An efficient synthetic route was developed and SAR studies toward activity and selectivity are described, leading to Lck inhibitors with enzymatic, cellular and in vivo potency.

Orally bioavailable cathepsin K inhibitors with pyrrolopyrimidine scaffold.

The recent emergence of osteoporosis as a major health threat in people of advanced age has intensified the search for novel and effective pharmacologic treatments. Given that bone resorption is exceeding bone formation, a reduction in bone mass leads to disease conditions including post-menopausal osteoporosis and tumor-induced osteolysis. Our efforts in this area have focused on the optimization of non-peptidic cathepsin K inhibitors for affinity and selectivity, from an heteroaromatic nitrile as a novel scaffold. This approach has resulted in the discovery of the potent and selective cathepsin K inhibitor, 44. The concentration of cathepsin K inhibitors, including compound 44, in the target tissues such as bone marrow cavity, were predictive parameters for antibone resorptive efficacy in vivo in the rat. The high level of distribution to the bone marrow was also observed for compounds containing pyrrolopyrimidines with novel spiro-structures as the P3 moiety. In a monkey study with the representative inhibitor 44, the antibone resorptive efficacy was detected 8 h after the compound administration. The efficacy persisted throughout the repeated treatment period of 14 days without any evidence for the development of tolerance. This article constitutes a near comprehensive review of the published scientific literature on small molecule non-peptidic inhibitors for cathepsin K developed by Novartis.

Small molecules for bone diseases.

IMPORTANCE OF THE FIELD: Bones play many roles in the body, providing structure, protecting organs, anchoring muscles and storing calcium. Over 100 million people worldwide suffer from bone diseases, mainly osteoporosis, cancer-related bone loss, osteoarthritis and inflammatory arthritis. Osteoporosis itself has no specific symptoms, and the main consequence is the increased risk of bone fractures. Therefore, the prevention of bone diseases is important to maintain the quality of life in the human society. However, treatment options are still insufficient. AREAS COVERED IN THIS REVIEW: This review article gives a summary of the low molecular mass modulators of bone diseases targets disclosed in patent applications and articles, mainly during the last 5 years. WHAT THE READER WILL GAIN: Readers will rapidly gain an overview of these modulators not only for historical targets, but also of emerging and re-visited targets. Readers will also be able to see the current research trend and the main players in this field. TAKE HOME MESSAGE: Drug discovery for bone diseases has made progress in the last years. The research area has dynamically shifted from historical targets (bisphosphonate, parathyroid hormone and calcitonin) to newly confirmed targets or targets re-visited which were biologically validated in the past. Cathepsin K inhibitors should be very close to launching in the market.

Knowledge-based virtual screening: application to the MDM4/p53 protein-protein interaction.

Chemogenomics knowledge-based drug discovery approaches aim to extract the knowledge gained from one target and to apply it for the discovery of ligands and hopefully drugs of a new target which is related to the parent target by homology or conserved molecular recognition. Herein, we demonstrate the potential of knowledge-based virtual screening by applying it to the MDM4-p53 protein-protein interaction where the MDM2-p53 protein-protein interaction constitutes the parent reference system; both systems are potentially relevant to cancer therapy. We show that a combination of virtual screening methods, including homology based similarity searching, QSAR (Quantitative Structure-Activity Relationship) methods, HTD (High Throughput Docking), and UNITY pharmacophore searching provide a successful approach to the discovery of inhibitors. The virtual screening hit list is of the magnitude of 50,000 compounds picked from the corporate compound library of approximately 1.2 million compounds. Emphasis is placed on the facts that such campaigns are only feasible because of the now existing HTCP (High throughput Cherry-Picking) automation systems in combination with robust MTS (Medium Throughput Screening) fluorescence-based assays. Given that the MDM2-p53 system constitutes the reference system, it is not surprising that significantly more and stronger hits are found for this interaction compared to the MDM4-p53 system. Novel, selective and dual hits are discovered for both systems. A hit rate analysis will be provided compared to the full HTS (High-throughput Screening).

Crystal Structures of Human MdmX (HdmX) in Complex with p53 Peptide Analogues Reveal Surprising Conformational Changes.

p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu(26), Trp(23), and Phe(19) are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 A of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp(23) of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an "open conformation" of Tyr(99) and unexpected "cross-talk" between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (K(d) values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2.

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.

Effect of cathepsin K inhibitors on bone resorption.

On the basis of the pyrrolopyrimidine core structure that was previously discovered, cathepsin K inhibitors having a spiro amine at the P3 have been explored to enhance the target, bone marrow, tissue distribution. Several spiro structures were identified with improved distribution toward bone marrow. The representative inhibitor 7 of this series revealed in vivo reduction in C-terminal telopeptide of type I collagen in rats and monkeys.