Artificial Intelligence for Drug Discovery: Are We There Yet?

Drug discovery is adapting to novel technologies such as data science, informatics, and artificial intelligence (AI) to accelerate effective treatment development while reducing costs and animal experiments. AI is transforming drug discovery, as indicated by increasing interest from investors, industrial and academic scientists, and legislators. Successful drug discovery requires optimizing properties related to pharmacodynamics, pharmacokinetics, and clinical outcomes. This review discusses the use of AI in the three pillars of drug discovery: diseases, targets, and therapeutic modalities, with a focus on small molecule drugs. AI technologies, such as generative chemistry, machine learning, and multi-property optimization, have enabled several compounds to enter clinical trials. The scientific community must carefully vet known information to address the reproducibility crisis. The full potential of AI in drug discovery can only be realized with sufficient ground truth and appropriate human intervention at later pipeline stages.Comment: 30 pages, 4 figures, 184 reference

SHP2 blockade sensitizes triple negative breast cancers to PI3K inhibition leading to metastatic shrinkage

Breast cancer is the most frequent and lethal cancer among women worldwide. A third of breast cancers can progress to metastasis, which remains the major cause of death in patients with solid tumors. Cells are regulated by numerous interconnected pathways, which can be dysregulated and results in uncontrolled proliferation. The phosphatidylinositol 3-kinase (PI3K) pathway is implicated in cellular growth, proliferation and survival, and is aberrantly activated in 70% of breast cancers. Despite the development of specific and effective drugs targeting the PI3K pathway, most clinical trial outcomes have been disappointing. In fact, mechanisms of resistance can short-circuit the efficacy of such inhibitors. Some of these mechanisms are receptor tyrosine kinase (RTK) driven, activating compensatory pathways and dramatically reducing the initial efficacy. Downstream of various active RTKs, Src-homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed protein-tyrosine phosphatase (PTP), transduces mitogenic, survival, cell-fate and/or migratory signals. Blockade of SHP2 has been shown to decrease breast tumor growth, progression and metastasis. Given that RTK-driven signaling pathways can overcome the effects of PI3K inhibition, and that SHP2 enhances signaling downstream of these receptors, we studied the effects of targeting PI3K and SHP2 simultaneously. In this study, we demonstrate a fundamental effect of PI3K/SHP2 dual-inhibition in triple-negative breast cancers (TNBCs), a very aggressive subtype associated with poor prognosis. Dual inhibition targeting PI3K and SHP2 appears to be more effective than single inhibitions by decreasing cell number in vitro and tumor volume in vivo, as well as increasing cancer cell apoptosis and improving animal survival. Mechanistically, SHP2 inhibition results in activation of the PI3K signaling and dependency on this pathway. We then assessed the effects of PI3K and/or SHP2 inhibitions on primary tumor growth, animal survival and lung metastases, a major metastatic site in breast cancer. While PI3K inhibition had no effects on primary tumor growth, it resulted in larger lung metastases in the neo-adjuvant setting. SHP2 inhibition decreased primary tumor growth as well as lung metastases. Both PI3K and SHP2 single treatment groups did not improve animal survival. In combination, PI3K/SHP2 dual-inhibition reduced synergistically primary tumor volumes, decreased lung metastases and increased animal survival. In the adjuvant setting, PI3K and SHP2 single inhibitions, as well as PI3K/SHP2 dual-inhibition, decreased lung metastases and increased animal survival. Despite the lack of lung metastases, concurrent PI3K/SHP2 blockade is not enough for complete metastasis regression. We demonstrated that liver metastases developed in parallel and have revealed to be insensitive to such inhibitions. We highlighted the discrepancy in RTK-dependences with lung metastases being PDGFRβ-dependant, while liver metastases are VEGFRs-dependent. Using a VEGFR/PDGFR inhibitor, we finally indicate that targeting PI3K/SHP2/VEGFR/PDGFR can further improve animal survival. The observations that pan-PI3K inhibition in the neo-adjuvant setting increases lung metastases in TNBC calls for caution when using such agents in the presence of the primary tumor. We have reported similar results using a dual-PI3K/mTOR inhibitor (Britschgi, Andraos et al. 2012). Our data provide a rationale for using pan-PI3K in combination with SHP2 inhibition to treat metastatic TNBC in the adjuvant setting and support further testing of this possibility. Moreover, we provide evidence that a triple therapy of PI3K, SHP2 and VEGFR/PDGFR inhibitors overcomes niche-dependent resistance and prolongs survival in preclinical models of TNBC

Applications of nuclear magnetic resonance spectroscopy: from drug discovery to protein structure and dynamics.

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors currently in clinical trials. This work combines screening of an FDA-approved drug panel and the identification of binders by protein-observed NMR. FDA-approved drugs salirasib and candesartan were identified as potent inhibitors in in vitro inhibition and migration assays while a weak inhibitor, olsalazine, was identified by NMR as the first small molecule inhibitor to directly bind PRL3. NMR was also used to validate the binding of additional compounds identified as experimental PRL3 inhibitors. Thienopyridone, a potent experimental inhibitor, did not show direct binding to PRL3 but instead inhibited phosphatase activity via redox mechanism. NMR also revealed that other experimental inhibitors did not engage PRL3. Thus, there remains a need to identify potent PRL3-directed inhibitors. Meanwhile, molecular modeling revealed a putative druggable site that has not been thoroughly explored before. The current study provides some scaffolds such as candesartan and particularly, olsalazine, the only binder identified, that could be the starting point of further drug discovery efforts, as well as a putative site that can be targeted in silico. MDMX, a negative regulator of p53, is another important therapeutic target in cancer, along with the homologous protein, MDM2. Inhibitors that block the MDM2-p53 interaction have been identified and despite similarities in the binding site of these homologous proteins, these inhibitors are ineffective against MDMX. It is hypothesized that the flexibility of MDMX contributes to this significant difference in response to inhibitors, despite comparable affinity to their endogenous target, p53. Examination of available inhibitor-bound structures of MDMX reveal a conserved pharmacophore but the structures adopt distinct conformations away from the binding site. This implies that global motions of the protein might contribute to molecular recognition. The conformational heterogeneity in MDMX was further confirmed by collecting residual dipolar couplings (RDCs). Further investigations on both MDMX and MDM2 are necessary to uncover whether the flexibility of MDMX contributes to the differential binding to inhibitors. Finally, NMR relaxation methods and state-of-the-art high-power Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion measurements, the first documented application on an enzyme, were used to characterize the solution dynamics of GMPK and the changes in dynamics upon GMP binding. Substrate binding resulted in restricting the amplitudes of motion for backbone amide bonds within the picosecond-nanosecond timescale. Meanwhile, CPMG showed dispersion in both in the absence and presence of GMP, such that substrate binding did not quench dynamics within the microsecond-millisecond timescale. Interestingly, more residues are observed to have dispersion in the bound form, some near the C-terminal of helix 3, which has previously been proposed to be involved in product release. Current studies show that substrate binding affect different timescales of protein motion. Future work shall follow how motions within different timescales are affected as GMPK processes its substrates – such as, for instance, binding of ATP analogs within the ATP binding site or simultaneous occupancy of both substrate binding pockets. This paves the way for a complete picture of the relationship of function and dynamics in the conformational enzymatic cycle of a bi-substrate enzyme using GMPK as a model. The current work illustrates some of the diverse applications of NMR on three unique systems that are also drug targets. Information collected here can be leveraged on future structure and dynamics studies as well as drug discovery efforts targeting any of these proteins

Therapeutic targeting of p90 ribosomal S6 kinase

The Serine/Threonine protein kinase family, p90 ribosomal S6 kinases (RSK) are downstream effectors of extracellular signal regulated kinase 1/2 (ERK1/2) and are activated in response to tyrosine kinase receptor or G-protein coupled receptor signaling. RSK contains two distinct kinase domains, an N-terminal kinase (NTKD) and a C-terminal kinase (CTKD). The sole function of the CTKD is to aid in the activation of the NTKD, which is responsible for substrate phosphorylation. RSK regulates various homeostatic processes including those involved in transcription, translation and ribosome biogenesis, proliferation and survival, cytoskeleton, nutrient sensing, excitation and inflammation. RSK also acts as a major negative regulator of ERK1/2 signaling. RSK is associated with numerous cancers and has been primarily studied in the context of transformation and metastasis. The development of specific RSK inhibitors as cancer therapeutics has lagged behind that of other members of the mitogen-activated protein kinase signaling pathway. Importantly, a pan-RSK inhibitor, PMD-026, is currently in phase I/1b clinical trials for metastatic breast cancer. However, there are four members of the RSK family, which have overlapping and distinct functions that can vary in a tissue specific manner. Thus, a problem for transitioning a RSK inhibitor to the clinic may be the necessity to develop isoform specific inhibitors, which will be challenging as the NTKDs are very similar to each other. CTKD inhibitors have limited use as therapeutics as they are not able to inhibit the activity of the NTKD but could be used in the development of proteolysis-targeting chimeras

ELUCIDATING THE ROLE OF THE TYROSINE PHOSPHATASE, SHP-2, IN REGULATION OF PD-L1 EXPRESSION IN NON-SMALL LUNG CANCER USING BOTH BIOCHEMICAL ANALYSES AND REAL-WORLD GENOMIC INFORMATION

Immune checkpoint inhibitors (ICIs), especially those that target programmed cell death protein 1 (PD-1) and programmed cell death ligand-1 (PD-L1), have been shown to provide substantial clinical benefit in many patients with non-small cell lung cancer (NSCLC). While these therapeutic agents can be highly effective in the correct context, the biological systems that malignant cells draft from normal activities of the cell are poorly characterized. Tumor cell-specific expression of PD-L1 is likely important for clinical benefit from PD-1 and PD-L1 inhibitors. It is known that PD-L1 is inappropriately expressed in many cancers harboring mutations in the RAS family of genes. The KRAS gene is mutated in as many as 30% of NSCLC tumor and drives tumor proliferation. Because there are no FDA-approved KRAS-targeting agents available for NSCLC patients, ICI therapy has been used in patients with tumors harboring mutations in the KRAS gene with clinical success. However, utilization of these therapies will remain hindered until there is a more complete understanding of the mechanisms governing the expression of targets of ICIs, specifically of PD-L1. The work in this dissertation explores the role of the tyrosine phosphatase, SHP-2. SHP-2 has been scrutinized as an important signaling molecule in a variety of cancers that links the activity of several signaling cascades as a regulator of KRAS, resulting in the clinical development of inhibitors of SHP-2. The work encompassed in these studies takes two complementary approaches to explore the role of SHP-2 in control of PD-L1 expression. First, publicly available real-world genomic information was used to establish a connection between the activity and/or expression of SHP-2 and PD-L1 in tumors and how expression relates to response to ICI therapy. Second, this work further sought to elucidate the molecular mechanism by which SHP-2 impacts the expression of PD-L1 in an NSCLC cell line model system. From these investigations, this work established that SHP-2 and PD-L1 have an expression relationship in clinical samples that may impact response to ICI therapies and experimentally identified a possible mechanism by which SHP-2 impacts PD-L1 expression in NSCLC

Inhibition of SHP1 and SHP2 as a molecular targeted therapy against myeloid leukaemias

[EN]Haematopoiesis is a very relevant differentiation process in adult humans where a multipotent cell, the haematopoietic stem cell (HSC), generates a widely varied, fully differentiated progeny, with immune defence, nutrient exchange and volume homeostasis functions. The regulatory cues governing the biology of HSCs must be tightly regulated in order to ensure their own self-renewal, as well as the proper turnover of differentiated cells. These signals are provided by the surrounding environment, known as niche, integrated by both haematopoietic and non-haematopoietic cells. The disruption of this fine equilibrium by alteration of either the external signals or their intracellular transduction in haematopoietic stem and progenitor cells (HSPCs) leads to the development of haematologic malignancies, including leukaemia. Two important blood disorders affecting the myeloid lineage are acute and chronic myeloid leukaemia (AML and CML, respectively). They are especially recurrent among the elderly, with a median age at diagnosis of 65 years for CML and 70 for AML, a fact to be considered due to the increasing life expectancy in Western countries. AML is a highly heterogeneous and aggressive disease with poor prognosis and, in general, no significant therapeutic improvements beyond chemotherapy over the last four decades. An exception to this scenario is the treatment of acute promyelocytic leukaemia (APL), which is highly responsive to pro-differentiative therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Unlike AML, CML is highly homogeneous in terms of molecular biology, with the expression of the fusion oncokinase breakpoint cluster region-ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL) as the main pathogenic driver. In the early 2000s, the clinical use of tyrosine kinase inhibitors (TKI) targeting this protein revolutionised the management of CML due to great improvements in treatment response and survival rates. However, this disease remains challenging in particular cases. Despite its heterogeneous nature, AML displays a differentiation blockage as a hallmark. This feature, together with the example of the differentiation-based APL treatment, has prompted the development of an important line of research focusing on the molecular mechanisms governing cell differentiation during haematopoiesis. This knowledge would lead to a better understanding of the dysregulated processes leading to pathogenesis and their subsequent pharmacological targeting to treat the disease. In line with this, the present work sought to assess in detail the involvement of SRC homology 2 domain containing protein tyrosine phosphatases 1 (SHP1) and 2 (SHP2) in the differentiation of leukaemic cells and the potential of these molecules as pharmacological targets for AML. Herein, it was demonstrated the cooperative function of both phosphatases in phorbol ester-induced cell differentiation, with an enhanced differentiated phenotype of cells subjected to simultaneous downregulation of these proteins. In addition, the kinase SRC was identified as a downstream target of SHP2 in this process, which appeared to influence the extent of the differentiation stimulus triggered by phorbol 12-myristate-13-acetate (PMA). Besides, the role of both phosphatases on cell differentiation showed to be partially overlapping through the regulation of β-catenin protein levels. Based on this evidence, the chemical inhibitor of SHP1 and SHP2 NSC 87877 (NSC) was successfully tested to boost the differentiation-inducing effect of phorbol esters in the AML cell line HL-60. Moreover, this compound synergised with the phorbol ester 13-O-acetyl-12-deoxyphorbol or prostratin (PRS) to prevent proliferation of not only in HL-60 cells, but also additional cell lines used as AML models (NB-4, OCI-AML2 and THP-1). Most importantly, the anti-leukaemic activity of this combination was corroborated in vivo with a xenograft mouse model and in primary cells from AML patients ex vivo. On the other hand, the management of CML still requires further improvement, since the success of TKI relies on long-term administration to patients due to the existence of quiescent BCR-ABL independent leukaemic stem cells (LSCs). This is associated with intolerances in patients and great costs for national health systems. Moreover, selective pressure on leukaemic blasts can lead to the emergence of point mutations in BCR-ABL that confer acquired resistance to TKI. To overcome this issue, novel therapeutic approaches based on co-targeting BCR-ABL and other important contributors to CML pathogenesis are subject of intense research. Based on this idea and the involvement of SHP1 and SHP2 in the disease, NSC was tested in combination with TKI in different models of CML. This compound enhanced the effect of the TKI imatinib (IM) and nilotinib (NL) in different CML cell lines (K-562, KCL-22 and BV-173). Immunoblotting analyses allowed the observation of a strong decrease in β-catenin levels upon NSC treatment and a moderate downregulation of c-MYC induced by both IM and NSC. Further gene expression studies identified CCND1, CCND2, CDKN1C and TLE2 as targets of either one or both drugs that might mediate the anti-leukaemic activity of their combination. Additionally, NSC also displayed anti-proliferative potential in patient-derived induced pluripotent stem cells (iPSCs), intrinsically resistant to IM at moderate doses. Treatment of these cells with NSC exerted a dramatic cell cycle arrest concomitant with the downregulation of CTNNB1, encoding β-catenin, as well as CCND1 and CCND2. Additionally, some members of TLE family were modulated by this drug. In summary, the results described in the present work support a promising therapeutic potential of the chemical inhibitor NSC 87877 in different myeloid malignancies where SHP1 and SHP2 are deeply involved. Furthermore, some mechanistic insight on the molecules connected to these phosphatases in both disease biology and pharmacological mode of action of the inhibitor has been provided

PROTACs – A Novel and Rapidly Developing Field of Targeted Protein Degradation

There is a continued need for new technology and strategies for tackling cancer and other diseases, and within the current century a novel therapeutic strategy has emerged in the realm of targeted protein degradation called Proteolysis-Targeting Chimeras (PROTACs). This technology specifically targets and degrades disease-causing proteins via the ubiquitin-proteasome system, and has seen an explosion of research and intrigue in both academia and industry over the past two decades. The diversity of PROTAC classes based on the E3 ligase recruiting ligand and the target protein allows for a universal molecular structure that can be customized for a specific target and disease. While it is primarily heavily focused in the realm of cancer therapeutics, PROTACs have expanded into other diseases such as cardiovascular, neurodegenerative, and virus-caused diseases. The discovery of novel PROTAC designs also allows for the field to overcome its own shortcomings and develop into new directions. Overall, the intrigue of PROTAC technology’s ability to degrade ‘undruggable’ targets has driven the field of research to expand rapidly in the short time since its initial discovery and continued intense efforts will help further shape the field to transition into the clinical setting to benefit the world

The leptin receptor complex : heavier than expected?

Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body weight by balancing food intake and energy expenditure. However, this adipocyte-derived hormone also directs peripheral processes, including immunity, reproduction, and bone metabolism. Leptin, therefore, can act as a metabolic switch connecting the body's nutritional status to high energy consuming processes. We provide an extensive overview of current structural insights on the leptin-ObR interface and ObR activation, coupling to signaling pathways and their negative regulation, and leptin functioning under normal and pathophysiological conditions (obesity, autoimmunity, cancer,.). We also discuss possible cross-talk with other receptor systems on the receptor (extracellular) and signaling cascade (intracellular) levels