An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth.

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

New Improved cGMP Analogues to Target Rod Photoreceptor Degeneration.

Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3',5'-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its RP-phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer SP-CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP.

Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT-2216.

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

A p97/Valosin-Containing Protein Inhibitor Drug CB-5083 Has a Potent but Reversible Off-Target Effect on Phosphodiesterase-6.

CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

NMR in integrated biophysical drug discovery for RAS: past, present, and future.

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.

Discovery of Novel PDEδ Degraders for the Treatment of KRAS Mutant Colorectal Cancer.

KRAS-PDEδ protein-protein interaction represents an appealing target for cancer therapy. However, fast release of high-affinity inhibitors from PDEδ hampered drug binding affinity and antiproliferative activity. To overcome the limitations, the first proteolysis-targeting chimeric (PROTAC) small molecules targeting PDEδ were designed. By employment of PDEδ inhibitor deltazinone (2) and cereblon ligand pomalidomide (6), a series of potent PROTAC PDEδ degraders were obtained. The most promising compound 17f efficiently induced PDEδ degradation and demonstrated significantly improved antiproliferative potency in KRAS mutant SW480 cells. Compound 17f also achieved significant tumor growth inhibition in the SW480 colorectal cancer xenograft model. This proof-of-concept study provided a new strategy to validate the druggability of KRAS-PDEδ interaction and offered an effective lead compound for the treatment of KRAS mutant cancer.

EPHA2 feedback activation limits the response to PDEδ inhibition in KRAS-dependent cancer cells.

KRAS is one of the most important proto-oncogenes. Its mutations occur in almost all tumor types, and KRAS mutant cancer is still lack of effective therapy. Prenyl-binding protein phosphodiesterase-δ (PDEδ) is required for the plasma membrane association and subsequent activation of KRAS oncogenic signaling. Recently, targeting PDEδ has provided new promise for KRAS mutant tumors. However, the therapeutic potential of PDEδ inhibition remains obscure. In this study, we explored how PDEδ inhibition was responded in KRAS mutant cancer cells, and identified KRAS mutant subset responsive to PDEδ inhibition. We first performed siRNA screen of KRAS growth dependency of a small panel of human cancer lines, and identified a subset of KRAS mutant cancer cells that were highly dependent on KRAS signaling. Among these cells, only a fraction of KRAS-dependent cells responded to PDEδ depletion, though KRAS plasma membrane association was effectively impaired. We revealed that the persistent RAF/MEK/ERK signaling seemed responsible for the lack of response to PDEδ depletion. A kinase array further identified that the feedback activation of EPH receptor A2 (EPHA2) accounted for the compensatory activation of RAF/MEK/ERK signaling in these cells. Simultaneous inhibition of EPHA2 and PDEδ led to the growth inhibition of KRAS mutant cancer cells. Together, this study gains a better understanding of PDEδ-targeted therapeutic strategy and suggests the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.

Identification of a new inhibitor of KRAS-PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer.

Oncogenic KRAS is considered a promising target for anti-cancer therapy. However, direct pharmacological strategies targeting KRAS-driven cancers remained unavailable. The prenyl-binding protein PDEδ, a transporter of KRAS, has been identified as a potential target for pharmacological inhibitor by selectively binding to its prenyl-binding pocket, impairing oncogenic KRAS signaling pathway. Here, we discovered a novel PDEδ inhibitor (E)-N'-((3-(tert-butyl)-2-hydroxy-6,7,8,9-tetrahydrodibenzo[b,dfuran-1-yl)methylene)-2,4-dihydroxybenzohydrazide(NHTD) by using a high-throughput docking-based virtual screening approach. In vitro and in vivo studies demonstrated that NHTD suppressed proliferation, induced apoptosis and inhibited oncogenic K-RAS signaling pathways by disrupting KRAS-PDEδ interaction in nonsmall cell lung cancer (NSCLC) harboring KRAS mutations. NHTD redistributed the localization of KRAS to endomembranes by targeting the prenyl-binding pocket of PDEδ and exhibited the suppression of abnormal KRAS function. Importantly, NHTD prevented tumor growth in xenograft and KRAS mutant mouse model, which presents an effective strategy targeting KRAS-driven cancer.

PDEδ inhibition impedes the proliferation and survival of human colorectal cancer cell lines harboring oncogenic KRas.

Ras proteins, most notably KRas, are prevalent oncogenes in human cancer. Plasma membrane localization and thereby signaling of KRas is regulated by the prenyl-binding protein PDEδ. Recently, we have reported the specific anti-proliferative effects of PDEδ inhibition in KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, we investigated the proliferative dependence on the solubilizing activity of PDEδ of human colorectal cancer (CRC) cell lines with or without oncogenic KRas mutations. Our results show that genetic and pharmacologic interference with PDEδ specifically inhibits proliferation and survival of CRC cell lines harboring oncogenic KRas mutations whereas isogenic cell lines in which the KRas oncogene has been removed, or cell lines with oncogenic BRaf mutations or EGFR overexpression are not dependent on PDEδ. Pharmacological PDEδ inhibition is therefore a possible new avenue to target oncogenic KRas bearing CRC.

Treatment of Macular Degeneration with Sildenafil: Results of a Two-Year Trial.

OBJECTIVE: To evaluate PDE5/6 inhibition with sildenafil to reduce choroidal ischemia and treat age-related macular degeneration. METHODS: Sildenafil was prescribed to treat participants with macular degenerations or macular dystrophies measured by spectral-domain optical coherence tomography, color fundus photography, enhanced depth imaging, and best-corrected visual acuity. RESULTS: No change in calcified drusen was noted. Vitelliform-type soft drusen were not substantially changed. A participant with Best vitelliform macular dystrophy had a significant improvement in vision as well as in photoreceptor and ellipsoid layers. CONCLUSIONS: Our research supports sildenafil as a safe treatment for age-related and vitelliform macular degenerations. Thickened Bruch's membrane reduces the beneficial effect of perfusion increase, but all eyes appear to benefit from PDE6. Notably, maintenance or improvement in the photoreceptor layer may be the most significant result of sildenafil and is consistent with PDE6 inhibition. Thus, sil-denafil treatment of macular degeneration offers significant potential for vision retention and recovery.

Electrophysiological determination of phosphodiesterase-6 inhibitor inhibition constants in intact mouse retina.

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. PDEs control numerous cellular processes making them promising targets for the development of therapeutic agents. Unfortunately, many PDE inhibitor molecules are non-selective among PDE classes and efficient methods for quantitative studies on the isoform-specificity of PDE inhibitors in the natural environments of PDEs are unavailable. The PDE in photoreceptors, PDE6, mediates the conversion of photon information into electrical signals making the retina an exceptional model system for examinations of the pharmacological effects of PDE inhibitors on PDE6. Here we introduce electroretinography-based methods for determining the inhibition constants of PDE inhibitors towards the naturally occurring light-activated and spontaneously activated forms of PDE6. We compare our results to earlier biochemical determinations with trypsin-activated PDE6 with disintegrated PDE6 γ-subunit. The potencies of PDE inhibitors were determined by stimulating the photoreceptors of isolated mouse retinas with light and tracking the inhibitor-induced changes in their electrical responses. The methods were tested with three PDE inhibitors, 3-isobutyl-1-methylxanthine (IBMX), sildenafil, and zaprinast. The inhibition constants towards light-activated, spontaneously activated, and trypsin-activated PDE6 differed significantly from each other for sildenafil and zaprinast but were closely similar for IBMX. We hypothesize that this is due to the ability of the PDE6 γ-subunit to compete with sildenafil and zaprinast from the same binding sites near the catalytic domain of PDE6. The introduced methods are beneficial both for selecting potent molecules for PDE6 inhibition and for testing the drugs targeted at other PDE isoforms against adverse effects on visual function.

Discovery of Novel KRAS-PDEδ Inhibitors by Fragment-Based Drug Design.

Targeting KRAS-PDEδ protein-protein interactions with small molecules represents a promising opportunity for developing novel antitumor agents. However, current KRAS-PDEδ inhibitors are limited by poor cellular antitumor potency and the druggability of the target remains to be validated by new inhibitors. To tackle these challenges, herein, novel, highly potent KRAS-PDEδ inhibitors were identified by fragment-based drug design, providing promising lead compounds or chemical probes for investigating the biological functions and druggability of KRAS-PDEδ interaction.

Efforts to Develop KRAS Inhibitors.

The high prevalence of KRAS mutations in human cancers and the lack of effective treatments for patients ranks KRAS among the most highly sought-after targets for preclinical oncologists. Pharmaceutical companies and academic laboratories have tried for decades to identify small molecule inhibitors of oncogenic KRAS proteins, but little progress has been made and many have labeled KRAS undruggable. However, recent progress in in silico screening, fragment-based drug design, disulfide tethered screening, and some emerging themes in RAS biology have caused the field to reconsider previously held notions about targeting KRAS. This review will cover some of the historical efforts to identify RAS inhibitors, and some of the most promising efforts currently being pursued.

Structural Biology-Inspired Discovery of Novel KRAS-PDEδ Inhibitors.

Structural biology is a powerful tool for investigating the stereospecific interactions between a protein and its ligand. Herein, an unprecedented chiral binding pattern was observed for inhibitors of KRAS-PDEδ interactions. Virtual screening and X-ray crystallography studies revealed that two enantiomers of a racemic inhibitor could bind at different sites. Fragment-based drug design was used to identify highly potent PDEδ inhibitors that can be used as promising lead compounds for target validation and antitumor drug development.

Effects of Fullerene Derivatives on Activity of Ca2+-ATPase of the Sarcoplasmic Reticulum and cGMP Phosphodiesterase.

We studied the effects of new water-soluble polysubstituted fullerene C60 (PFD) derivatives on activity of Ca2+-Mg2+ ATPase of the sarcoplasmic reticulum and cGMP phosphodiesterase. All examined fullerene derivatives inhibited activity of both enzymes. For instance, PFD-I, PFD-II, PFD-III, PFD-V, PFD-IX, PFD-X, and PFD-XI in a concentration of 5×10-5 M completely inhibited hydrolytic and transport functions of Ca2+-ATPase. These compounds in a concentration of 5×10-6 suppressed active transport of calcium ions by 51±5, 77±8, 52±5, 52±5, 100±10, 80±8, and 100±10%, respectively, and inhibited ATP hydrolysis by 31±3, 78±8, 18±2, 29±3, 78±8, 63±7, and 73±9%, respectively, uncoupling the hydrolytic and transport functions of the enzyme. PFD-I noncompetitive and reversibly reduced activity of Ca2+-ATPase (Ki=2.3×10-6 M). All the studied fullerene derivatives (except for PFD-VII) inhibited cGMP phosphodiesterase by more than 80% in concentration of 10-4 M and higher and by more than 50% in concentration of 10-5 M. PFD-I is a non-competitive reversible inhibitor of cGMP phosphodiesterase (Ki=7×10-6 M). These results allow us to expect antimetastatic, antiaggregatory, antihypertensive and vasodilative activity of the studied compounds.

Structure-based development of PDEδ inhibitors.

The prenyl binding protein PDEδ enhances the diffusion of farnesylated Ras proteins in the cytosol, ultimately affecting their correct localization and signaling. This has turned PDEδ into a promising target to prevent oncogenic KRas signaling. In this review we summarize and describe the structure-guided-development of the three different PDEδ inhibitor chemotypes that have been documented so far. We also compare both their potency for binding to the PDEδ pocket and their in vivo efficiency in suppressing oncogenic KRas signaling, as a result of the inhibition of the PDEδ/KRas interaction.

Identification of pyrazolopyridazinones as PDEδ inhibitors.

The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.

Molecular Simulations of Solved Co-crystallized X-Ray Structures Identify Action Mechanisms of PDEδ Inhibitors.

PDEδ is a small protein that binds and controls the trafficking of RAS subfamily proteins. Its inhibition protects initiation of RAS signaling, and it is one of the common targets considered for oncological drug development. In this study, we used solved x-ray structures of inhibitor-bound PDEδ targets to investigate mechanisms of action of six independent all-atom MD simulations. An analysis of atomic simulations combined with the molecular mechanic-Poisson-Boltzmann solvent accessible surface area/generalized Born solvent accessible surface area calculations led to the identification of action mechanisms for a panel of novel PDEδ inhibitors. To the best of our knowledge, this study is one of the first in silico investigations on co-crystallized PDEδ protein. A detailed atomic-scale understanding of the molecular mechanism of PDEδ inhibition may assist in the design of novel PDEδ inhibitors. One of the most common side effects for diverse small molecules/kinase inhibitors is their off-target interactions with cardiac ion channels and human-ether-a-go-go channel specifically. Thus, all of the studied PDEδ inhibitors are also screened in silico at the central cavities of hERG1 potassium channels.

Design and synthesis of novel 5-(3,4,5-trimethoxybenzoyl)-4-aminopyrimidine derivatives as potent and selective phosphodiesterase 5 inhibitors: scaffold hopping using a pseudo-ring by intramolecular hydrogen bond formation.

5-(3,4,5-Trimethoxybenzoyl)-4-amimopyrimidine derivatives were found as a novel chemical class of potent and highly selective phosphodiesterase 5 inhibitors. A pseudo-ring formed by an intramolecular hydrogen bond constrained the conformation of 3-chloro-4-methoxybenzylamino and 3,4,5-trimethoxybenzoyl substituents and led to the discovery of T-6932 (19a) with a potent PDE5 inhibitory activity (IC50 = 0.13 nM) and a high selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2400). Further modification at the 2-position of T-6932 resulted in the finding of 26, which exhibited potent relaxant effects on isolated rabbit corpus cavernosum (EC30 = 11 nM) with a high PDE5 selectivity over PDE6 (IC50 ratio: PDE6/PDE5 = 2800).

Structure guided design and kinetic analysis of highly potent benzimidazole inhibitors targeting the PDEδ prenyl binding site.

K-Ras is one of the most frequently mutated signal transducing human oncogenes. Ras signaling activity requires correct cellular localization of the GTPase. The spatial organization of K-Ras is controlled by the prenyl binding protein PDEδ, which enhances Ras diffusion in the cytosol. Inhibition of the Ras-PDEδ interaction by small molecules impairs Ras localization and signaling. Here we describe in detail the identification and structure guided development of Ras-PDEδ inhibitors targeting the farnesyl binding pocket of PDEδ with nanomolar affinity. We report kinetic data that characterize the binding of the most potent small molecule ligands to PDEδ and prove their binding to endogenous PDEδ in cell lysates. The PDEδ inhibitors provide promising starting points for the establishment of new drug discovery programs aimed at cancers harboring oncogenic K-Ras.