ABSTRACT
Herpesvirus infections are ubiquitous, with over 95% of the adult population infected by at least one strain. While most of these infections resolve without treatment in healthy individuals, they can cause significant morbidity and mortality in immunocompromised, stem cell, or organ transplant patients. Current nucleoside standards of care provide meaningful benefit but are limited due to poor tolerability, resistance, and generally narrow spectrum of activity. Herpesviruses share a conserved DNA polymerase, the inhibition of which is validated as an effective strategy to disrupt viral replication. By utilizing a non-nucleoside inhibitor of the viral DNA polymerase, we sought to develop agents covering multiple herpesviruses (e.g., CMV, VZV, HSV1/2, EBV, and HHV6). Herein is described the invention of an oxazolidinone class of broad-spectrum non-nucleoside herpes antiviral inhibitors. A lead compound (42) with potent biochemical and broad-spectrum cellular activity was found to be efficacious in murine models against both HSV-1 and CMV infection.
ABSTRACT
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).
Subject(s)
Antiviral Agents , SARS-CoV-2 , Animals , Mice , SARS-CoV-2/drug effects , Humans , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/genetics , COVID-19 Drug Treatment , Protease Inhibitors/pharmacology , COVID-19/virology , Coronavirus Infections/drug therapy , Coronavirus Infections/virologyABSTRACT
As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.
Subject(s)
COVID-19 , Glutamine , Humans , Glutamine/chemistry , SARS-CoV-2 , Cysteine Endopeptidases/chemistry , Inventions , Protease Inhibitors/pharmacology , Amides , Antiviral Agents/pharmacology , Antiviral Agents/chemistryABSTRACT
Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.
ABSTRACT
Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.
Subject(s)
Anti-HIV Agents , HIV Integrase Inhibitors , HIV Integrase , HIV Integrase Inhibitors/pharmacology , HIV Integrase Inhibitors/chemistry , Allosteric Regulation , Catalytic Domain , HIV Integrase/metabolismABSTRACT
Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.
ABSTRACT
Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.
Subject(s)
Benzoxazoles/pharmacology , Dopaminergic Neurons/drug effects , Glucosylceramidase/genetics , Glucosyltransferases/antagonists & inhibitors , Glycosphingolipids/metabolism , Lysosomes/drug effects , Synucleinopathies/metabolism , alpha-Synuclein/drug effects , Animals , Dopaminergic Neurons/metabolism , In Vitro Techniques , Lysosomes/metabolism , Mice , Neurons/drug effects , Neurons/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Primary Cell Culture , Protein Aggregates , Rats , Synucleinopathies/genetics , alpha-Synuclein/metabolismABSTRACT
All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/drug effects , DNA-Directed DNA Polymerase/genetics , Herpesviridae/drug effects , Herpesviridae/enzymology , Antiviral Agents/pharmacology , Binding Sites , DNA-Directed DNA Polymerase/metabolism , Drug Resistance, Viral/drug effects , Exodeoxyribonucleases , Nucleotides , Quinolines/pharmacology , Viral Proteins , Virus ReplicationABSTRACT
A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.
ABSTRACT
Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.
Subject(s)
Allosteric Regulation/drug effects , HIV Infections/drug therapy , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/drug effects , Cell Line , Drug Discovery , HIV Infections/metabolism , HIV Infections/virology , HIV-1/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Protein Interaction Maps/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor, trkA/antagonists & inhibitors , Receptor, trkA/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Kinetics , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Models, Molecular , Protein Conformation , Protein Kinase Inhibitors/chemical synthesis , Receptor, trkA/genetics , Receptor, trkB/antagonists & inhibitors , Receptor, trkB/chemistry , Receptor, trkB/genetics , Receptor, trkC/antagonists & inhibitors , Receptor, trkC/chemistry , Receptor, trkC/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Recombinant Proteins/genetics , Structure-Activity Relationship , Surface Plasmon ResonanceABSTRACT
With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/enzymology , Macrocyclic Compounds/pharmacology , Quinazolinones/pharmacology , Sulfones/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Crystallography, X-Ray , Drug Discovery , Hepacivirus/drug effects , Hepacivirus/genetics , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacokinetics , Models, Molecular , Mutation , Quinazolinones/chemistry , Quinazolinones/pharmacokinetics , Rats , Sulfones/pharmacokinetics , Viral Nonstructural Proteins/geneticsABSTRACT
The viral transactivator protein (Tat) plays an essential role in the replication of human immunodeficiency type 1 virus (HIV-1) by recruiting the host positive transcription elongation factor (pTEFb) to the RNA polymerase II transcription machinery to enable an efficient HIV-1 RNA elongation process. Blockade of the interaction between Tat and pTEFb represents a novel strategy for developing a new class of antiviral agents. In this study, we developed a homogeneous assay in AlphaLISA (amplified luminescent proximity homogeneous assay) format using His-tagged pTEFb and biotinylated Tat to monitor the interaction between Tat and pTEFb. On optimizing the assay conditions, the signal-to-background ratio was found to be greater than 10-fold. The assay was validated with untagged Tat and peptides known to compete with Tat for pTEFb binding. The Z' of the assay is greater than 0.5, indicating that the assay is robust and can be easily adapted to a high-throughput screening format. Furthermore, the affinity between Tat and pTEFb was determined to be approximately 20 pM, and only 7% of purified Tat was found to be active in forming tertiary complex with pTEFb. Development of this assay should facilitate the discovery of a new class of antiviral agents providing HIV-1 patients with broader treatment choices.
Subject(s)
HIV-1/chemistry , Luminescent Measurements/methods , Multiprotein Complexes/chemistry , Positive Transcriptional Elongation Factor B/chemistry , tat Gene Products, Human Immunodeficiency Virus/chemistry , Animals , HIV-1/genetics , HIV-1/metabolism , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Positive Transcriptional Elongation Factor B/genetics , Positive Transcriptional Elongation Factor B/metabolism , RNA, Viral/biosynthesis , RNA, Viral/chemistry , RNA, Viral/genetics , Sf9 Cells , Spodoptera , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Hepacivirus/enzymology , Macrocyclic Compounds/chemistry , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Binding Sites , Carrier Proteins/metabolism , Catalytic Domain , Cyclization , Genotype , Half-Life , Hepacivirus/genetics , Intracellular Signaling Peptides and Proteins , Kinetics , Liver/metabolism , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacokinetics , Molecular Docking Simulation , Mutation , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacokinetics , Rats , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Hepacivirus/enzymology , Macrocyclic Compounds/chemistry , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Carrier Proteins/metabolism , Cyclization , Genotype , Half-Life , Hepacivirus/genetics , Intracellular Signaling Peptides and Proteins , Kinetics , Liver/metabolism , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/pharmacokinetics , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacokinetics , Quinolines/chemistry , Rats , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.
Subject(s)
Hepacivirus/drug effects , Protease Inhibitors/pharmacology , Quinoxalines/pharmacology , Quinoxalines/pharmacokinetics , Viral Nonstructural Proteins/antagonists & inhibitors , Amides , Animals , Antiviral Agents/pharmacology , Carbamates , Cyclopropanes , Dogs , Drug Resistance, Viral , Genotype , Hepacivirus/enzymology , Hepacivirus/genetics , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/virology , Liver/drug effects , Pan troglodytes , Quinoxalines/metabolism , Rats , Sulfonamides , Viral Load/drug effectsABSTRACT
A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.
ABSTRACT
In the present paper, design, synthesis, X-ray crystallographic analysis, and HIV-1 protease inhibitory activities of a novel class of compounds are disclosed. Compounds 28-30, 32, 35, and 40 were synthesized and found to be inhibitors of the HIV-1 protease. The crucial step in their synthesis involved an unusual endo radical cyclization process. Absolute stereochemistry of the three asymmetric centers in the above compounds have been established to be (4S,2'R,3'S) for optimal potency. X-ray crystallographic analysis has been used to determine the binding mode of the inhibitors to the HIV-1 protease.
Subject(s)
Carbamates/chemical synthesis , HIV Protease Inhibitors/chemical synthesis , HIV Protease/chemistry , Models, Molecular , Thiazepines/chemical synthesis , Carbamates/chemistry , Crystallography, X-Ray , Drug Design , HIV Protease Inhibitors/chemistry , Molecular Structure , Protein Binding , Protein Conformation , Stereoisomerism , Structure-Activity Relationship , Thiazepines/chemistryABSTRACT
A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.