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The melanocortin-3 receptor (MC3R) regulates GABA release from agouti-related protein (AgRP) nerve terminals and thus tonically suppresses multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of the MC3R and the melanocortin system in regulating the response to various anorexigenic agents. The genetic deletion or pharmacological inhibition of the MC3R, or subthreshold doses of an MC4R agonist, improved the dose responsiveness to glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss. An enhanced anorectic response to the acute satiety factors peptide YY (PYY3-36) and cholecystokinin (CCK) and the long-term adipostatic factor leptin demonstrated that increased sensitivity to anorectic agents was a generalized result of MC3R antagonism. We observed enhanced neuronal activation in multiple hypothalamic nuclei using Fos IHC following low-dose liraglutide in MC3R-KO mice (Mc3r-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits that control multiple aspects of feeding behavior. The enhanced anorectic response in Mc3r-/- mice after administration of GLP1 analogs was also independent of the incretin effects and malaise induced by GLP1 receptor (GLP1R) analogs, suggesting that MC3R antagonists or MC4R agonists may have value in enhancing the dose-response range of obesity therapeutics.

We employed a structural bioinformatics approach to develop novel peptides with predicted affinity to the binding site for negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5). Primary screening in zebrafish ( Danio rerio ) revealed a stimulatory effect of two peptides, LCGM-10 and LCGM-15. Target validation studies using calcium ion flux imaging and a luciferase reporter assay confirmed mGluR5 as the target. LCGM-10 showed greater potency than LCGM-15; it was comparable to that of the mGluR5 NAM 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rodent behavioral screening in the open field and elevated plus maze revealed increased locomotor activity in both tests after acute LCGM-10 treatment, supported by further analysis of home cage spontaneous locomotor activity (SLA). The stimulating effect of a single LCGM-10 administration on SLA was evident up to 60 min after administration and was not accompanied by hypokinetic rebound observed for caffeine. According to our results, LCGM-10 has therapeutic potential to treat hypo- and dyskinesias of various etiologies. Further investigation of LCGM-10 effects in the delay discounting model of impulsive choice in rats revealed reduced trait impulsivity after single and chronic administrations, suggesting potential implication for attention deficit hyperactivity disorder, obsessive compulsive disorder, and addictions., Competing Interests: AM, IS, VP, ID, NM, VG, and GB are employed by Lactocore Inc. TS is a founder of Maestro Therapeutics and a consultant for Lactocore Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Malyshev, Pavshintcev, Mitkin, Sukhanova, Gedzun, Zlobin, Doronin, Babkin and Sawyer.)

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif., (© 2024. Merck & Co., Inc., Rahway, NJ, USA and its affiliates and Tsz Ying Yuen, Diana Spiegelberg, Pietro G. Aronica, Yee Hwee Lim, Marika Nestor, Chandra S. Verma, David P. Lane, Srinivasaraghavan Kannan, Christopher J. Brown, Charles W. Johannes.)

Setmelanotide (Imcivree™) was developed as a daily injectable therapeutic peptide for the treatment of rare forms of syndromic obesity, such as POMC deficiency and leptin receptor deficiency. The important option of poly(lactic-co-glycolic acid) (PLGA) controlled release microspheres has become more attractive for this class of drugs upon the discovery that net positively charged peptides can be remote-loaded rapidly from aqueous peptide solution into blank microspheres at high loading and encapsulation efficiency. Here we sought to remote-load setmelanotide in PLGA microspheres and examine its potential for long-term controlled release and body weight control. The influence of PLGA microsphere porosity was investigated with respect to morphology, drug loading, and in vitro release profiles. Increased density of the microspheres inhibited the progress of encapsulation of the dicationic peptide. A diet-induced obese murine model was then used to determine the pharmacokinetic profile and to evaluate long-term efficacy of an optimal formulation. Remote loaded PLGA formulations encapsulated setmelanotide as high as ∼63% (∼6.3% w/w loading) and exhibited slow and continuous peptide release over ∼6 weeks in vitro largely independent of microsphere porosity. The obtained in vivo release pattern from deconvolution of the pharmacokinetics after subcutaneous microsphere injection was consistent with the in vitro release profile but with a lower initial burst release and overall slightly faster release rate. After a single injection of remote-loaded setmelanotide, continuous long-term inhibition of food intake and body weight control was observed over 17 and 30 days, respectively. The improvement in body weight control over drug-free microsphere vehicle-treated control groups matched the observed PK profile. This study provides the first report of long-acting release formulation for 1-month controlled release of setmelanotide and body weight control in a diet induced obese murine model, and supports the further development of long-acting treatment options for obese patients., Competing Interests: Declaration of Competing Interest RDC, TKS, and the University of Michigan have equity in Courage Therapeutics, and RDC serves on the board of the company., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We report the discovery of sulanemadlin (ALRN-6924), the first cell-permeating, stabilized α-helical peptide to enter clinical trials. ALRN-6924 is a "stapled peptide" that mimics the N-terminal domain of the p53 tumor suppressor protein. It binds with high affinity to both MDM2 and MDMX (also known as MDM4), the endogenous inhibitors of p53, to activate p53 signaling in cells having a non-mutant, or wild-type TP53 genotype ( TP53 -WT). Iterative structure-activity optimization endowed ALRN-6924 with favorable cell permeability, solubility, and pharmacokinetic and safety profiles. Intracellular proteolysis of ALRN-6924 forms a long-acting active metabolite with potent MDM2 and MDMX binding affinity and slow dissociation kinetics. At high doses, ALRN-6924 exhibits on-mechanism anticancer activity in TP53 -WT tumor models. At lower doses, ALRN-6924 transiently arrests the cell cycle in healthy tissues to protect them from chemotherapy without protecting the TP53 -mutant cancer cells. These results support the continued clinical evaluation of ALRN-6924 as an anticancer and chemoprotection agent.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2') 7 ), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2') 7 -containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Purpose: To develop a novel, target agnostic liposome click membrane permeability assay (LCMPA) using liposome encapsulating copper free click reagent dibenzo cyclooctyne biotin (DBCO-Biotin) to conjugate azido modified peptides that may effectively translocate from extravesicular space into the liposome lumen., Method: DBCO-Biotin liposomes were prepared with egg phosphatidylcholine and cholesterol by lipid film rehydration, freeze/thaw followed by extrusion. Size of DBCO-Biotin liposomes were characterized with dynamic light scattering., Results: The permeable peptides representing energy independent mechanism of permeability showed higher biotinylation in LCMPA. Individual peptide permeability results from LCMPA correlated well with shifts in potency in cellular versus biochemical assays (i.e., cellular/ biochemical ratio) demonstrating quantitative correlation to intracellular barrier in intact cells., Conclusion: The study provides a novel membrane permeability assay that has potential to evaluate energy independent transport of diverse peptides.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future., (Copyright © 2020. Published by Elsevier Inc.)

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein-protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from d-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-d α-helical stapled and stitched peptides. As a template, we used a recently reported all d-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-d-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all d-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity., (This journal is © The Royal Society of Chemistry 2020.)

Nonspecific promiscuous compounds can mislead researchers and waste significant resources. This phenomenon, though well-documented for small molecules, has not been widely explored for the peptide modality. Here we demonstrate that two purported peptide-based KRas inhibitors, SAH-SOS1 A and cyclorasin 9A5, exemplify false-positive molecules-in terms of both their binding affinities and cellular activities. Through multiple gold-standard biophysical techniques, we unambiguously show that both peptides lack specific binding to KRas and instead induce protein unfolding. Although these peptides inhibited cellular proliferation, the activities appeared to be off-target on the basis of a counterscreen with KRas-independent cell lines. We further demonstrate that their cellular activities are derived from membrane disruption. Accordingly, we propose that to de-risk false-positive molecules, orthogonal binding assays and cellular counterscreens are indispensable., Competing Interests: The authors declare the following competing financial interest(s): S.K. and C.S.V. are founders of Sinopsee Therapeutics; the current work has no conflicts.

There is interest in peptide drug design, especially for targeting intracellular protein-protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled α-helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design.

Purpose: In this study we evaluated the utility of in-vitro screening tools for predicting the in-vivo behavior of six cyclic peptides with different solubility and permeability properties (BCS class II and III), intended for oral delivery in presence of permeation enhancer Labrasol., Methods: An in vitro flux assay was used to assess peptide permeation across a biomimetic, lipid-based membrane and in vivo studies in rats were used to determine oral peptide bioavailability in the presence of Labrasol., Results: The in vitro flux was significantly increased for BCS class III peptides, while it significantly decreased or remained unchanged for BCS class II peptides with increasing Labrasol concentrations. The different flux responses were attributed to the combination of reduced effective free peptide concentration and increased membrane permeability in the presence of Labrasol. In vivo studies in male Wistar-Hans rats indicated improved oral bioavailability at different extents for all peptides in presence of Labrasol. On comparing the in vitro and in vivo data, a potential direct correlation for BCS class III peptides was seen but not for BCS class II peptides, due to lower free concentrations of peptides in this class., Conclusion: This study assessed the utility of in vitro screening tools for selecting peptides and permeation excipients early in drug product development. Graphical Abstract Graphical Abstract and Figure 1 contains small text.Graphical Abstract text is made larger. The Figure 1 text cannot be made larger.

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe 3 , Trp 7 , and Cba 10 ) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically 'break' right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe 3 and Cba 10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp 7 to d-Trp 7 ) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp 7 -modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R 8 4 to Aib 4 and S 5 11 to Aib 11 ) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac-Leu-Thr-Phe-Aib-Glu-Tyr-Trp-Gln-Leu-Cba-Aib-Ser-Ala-Ala-NH 2 exhibited high-affinity target binding (Mdm2 K d = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu 5 , d-Gln 8 , and d-Leu 9 ) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp 7 -modification, a highly potent ATSP-7041 analog (Mdm2 K d = 30 nM; cellular EC 50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

Ni/photoredox (4DPAIPN) dual catalysis enabled challenging peptide C(sp 2 )-O coupling reactions. Successful cross-coupling reactions were demonstrated with highly functionalized alcohols including side chains of amino acids ( i.e. , serine, threonine, tyrosine), trans -4-hydroxy-l-proline, alkyl alcohols, alkynylated alcohols, and carbohydrates. Coupling reactions between bromobenzoyl-capped peptides containing various side chains and either a protected serine building block or a serine-containing dipeptide also proceeded efficiently. Chemoselective C-O coupling (over C-N) was achieved in intermolecular reactions in the presence of a C-terminal primary amide. Furthermore, by judicious structural design in combination with computational modeling, we demonstrated side chain-to-tail macrocyclization of peptides containing a β-turn motif via C-O coupling. The methodology developed in this work brings new opportunities for late-stage diversification of complex linear and macrocyclic peptides.

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development., (Copyright © 2018. Published by Elsevier Ltd.)

Traditionally, computing the binding affinities of proteins to even relatively small and rigid ligands by free-energy methods has been challenging due to large computational costs and significant errors. Here, we apply a new molecular simulation acceleration method called MELD (Modeling by Employing Limited Data) to study the binding of stapled α-helical peptides to the MDM2 and MDMX proteins. We employ free-energy-based molecular dynamics simulations (MELD-MD) to identify binding poses and calculate binding affinities. Even though stapled peptides are larger and more complex than most protein ligands, the MELD-MD simulations can identify relevant binding poses and compute relative binding affinities. MELD-MD appears to be a promising method for computing the binding properties of peptide ligands with proteins.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

In this issue of Expert Opinion on Investigational Drugs, several protein kinases families and pathways underlying cancer and other diseases are reviewed and several small molecule inhibitors that are in clinical trials are further described. Highlights of these reviews and drug evaluations are summarized in this editorial.

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein-protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled alpha-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted alpha-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha-helical stability. These simulations provide new insights into the design of alpha-helical stapled peptides and the development of potent inhibitors of alpha-helical protein-protein interfaces.

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukaemia. The second generation BCR/ABL inhibitors nilotinib and dasatinib effectively inhibit most imatinib resistance variants, but are ineffective against the gatekeeper mutant, T315I. Gatekeeper mutation activates the kinase by stabilizing the hydrophobic spine. Here, we describe that the rationally designed compound AP24163 can inhibit native and gatekeeper mutants of the BCR/ABL kinase. Structural modelling suggests that AP24163 affects the flexibility of the P-loop and destabilizes the active conformation by disrupting the hydrophobic spine. In vitro screening for drug resistance identified clones with compound mutations involving both the P-loop and T315I. Our studies provide structural insights for the design of inhibitors against the gatekeeper mutant and suggest that up-front combination therapy may be required to prevent the emergence of compound-resistant mutations.

Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by two such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy. Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency (IC50 = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed therapies for chronic myeloid leukemia.

Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Here, we describe structure activity relationships of a novel series of carbon-linked, 2-substituted purines that led to the identification of AP23451 as a potent inhibitor of Src tyrosine kinase with antiresorptive activity in vivo. AP23451 features the use of an arylphosphinylmethylphosphinic acid moiety which confers bone-targeting properties to the molecule, thereby increasing local concentrations of the inhibitor to actively resorbing osteoclasts at the bone interface. AP23451 exhibited an IC50 = 68 nm against Src kinase; an X-ray crystal structure of the molecule complexed with Src detailed the molecular interactions responsible for its Src inhibition. In vivo, AP23451 demonstrated a dose-dependent decrease in PTH-induced hypercalcemia. Moreover, AP23517, a structurally and biochemically similar molecule with comparable activity (IC50 = 73 nm) except devoid of the bone-targeting element, demonstrated significantly reduced in vivo efficacy, suggesting that Src activity was necessary but not sufficient for in vivo activity in this series of compounds.

Cell adhesion receptors, referred to as integrins, are recognized as key regulators of cellular processes including growth and differentiation. Integrins communicate with growth factor receptors (GFRs) to control specific cellular responses to stimuli originating in the extracellular environment. In this article, we review the role of integrins as molecular switches that modulate GFR activation and specificity. We also examine the reciprocal modulation of integrin functions by GFRs and the mechanisms through which those actions are fine-tuned., (2007 Wiley-Liss, Inc.)

Src tyrosine kinase was the first gene product shown to have an essential function in bone using recombinant DNA technology after its expression was knocked out in mice approximately 15 years ago. Since then, our understanding of the regulation of bone catabolism has advanced significantly with the identification of other key enzymes that regulate osteoclast formation, activation, and survival after their knockout in mice or recognition of mutations in them in humans. This led to the discovery or development of specific inhibitors of some of these key enzymes, including Src, as proof-of-concept lead compounds or potential clinical candidates for the prevention of diseases associated with increased bone resorption, such as osteoporosis and metastatic bone disease. Although bisphosphonates have been prescribed with proven and improving efficacy for the prevention of bone loss for >30 years, adverse effects, such as upper gastrointestinal tract symptoms, and the requirement to take them at least 2 hours before food have limited patient compliance. Thus, with growing knowledge of the pathways regulating osteoclast function and the appreciation that some of these are active also in tumor cells, drug companies have made efforts to identify small-molecular lead compounds for development into new therapeutic agents for the prevention of bone loss with efficacy that matches or supersedes that of bisphosphonates. In this article, we review our current understanding of the signaling pathways that regulate osteoclast formation, activation, and survival with specific reference to the role of Src tyrosine kinase and downstream signaling and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential adverse effects on other cells.