Covalent Inhibition of Wild-Type HIV-1 Reverse Transcriptase Using a Fluorosulfate Warhead

Article abstract of DOI:10.1021/acsmedchemlett.0c00612

Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether non-nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC50 values for in vitro inhibition of WT RT and EC50 values for cytopathic protection of HIV-1-infected human T-cells in the 5-320 nM range.

Full text of DOI:10.1021/acsmedchemlett.0c00612

pubs.acs.org/acsmedchemlett
Letter
Covalent Inhibition of Wild-Type HIV‑1 Reverse Transcriptase Using
a Fluorosulfate Warhead
Joseph A. Ippolito,∥ Haichan Niu,∥ Nicole Bertoletti,∥ Zachary J. Carter, Shengyan Jin,
́
Krasimir A. Spasov, Jose A. Cisneros, Margarita Valhondo, Kara J. Cutrona, Karen S. Anderson,*
and William L. Jorgensen*
Cite This: ACS Med. Chem. Lett. 2021, 12, 249−255
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Covalent inhibitors of wild-type HIV-1 reverse transcriptase
(CRTIs) are reported. Three compounds derived from catechol diether non-
nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are
demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal
structures for complexes of the CRTIs with the enzyme are provided,
which fully demonstrate the covalent attachment, and confirmation is
provided by appropriate mass shifts in ESI-TOF mass spectra. The three
CRTIs and six noncovalent analogues are found to be potent inhibitors with
both IC50 values for in vitro inhibition of WT RT and EC50 values for
cytopathic protection of HIV-1-infected human T-cells in the 5−320 nM
range.
KEYWORDS: anti-HIV agents, reverse transcriptase inhibitors, covalent inhibitors, fluorosulfates
he HIV/AIDS crisis continues as a major pandemic with
worldwide statistics of 37 million people infected, 2
investigated approaches are slow-release formulations in
injected nanoparticles or in subcutaneous polymer-based
implants. A drawback of the former approach is the difficulty
in response to possible adverse effects, while implants, though
more difficult to administer, can be removed. Our laboratories
have pursued development of improved NNRTIs with high-
potency against with-type HIV-1 and clinically important
variants;7−11 we have reported compounds with excellent
pharmacokinetics,12−14 and representative ones have been
demonstrated to sustain high plasma levels and efficacy for
more than 3 weeks in humanized mice after a single injection
of a nanoformulation.13,14
We have also been exploring a new class of anti-HIV agents,
covalent inhibitors of HIV-1 reverse transcriptase (CRTIs).
The initial efforts successfully targeted clinically problematic
Tyr181Cys RT variants.15 Conclusive evidence for the
covalent modification of Cys181 included results from mass
spectrometry (MS), protein crystallography, and antiviral
activity in infected human T-cell assays. The CRTIs were
shown to be selective for Cys181 and have lower cytotoxicity
T
million new infections, and 1 million deaths in 2017, roughly
equally split between men and women.1 Considering the rate
of new infections and that 40% of the infected people are not
receiving drug treatment, the grave problems will continue at
high levels for many years. Nevertheless, highly active
antiretroviral therapy (HAART) has had significant impact
with the annual deaths reduced by half from the peak of 1.95
million in 2006.1 Drugs have been discovered in multiple
classes, with nucleoside reverse transcriptase inhibitors
(NRTIs), non-nucleoside RTIs (NNRTIs), protease inhibitors
(PIs), and integrase inhibitors (INTIs) being the most widely
used, typically in three-drug combinations.2 The problems with
pill-burden have much improved with the introduction of daily
single-tablet regimens (STRs), which have predominantly
featured coformulation of two NRTIs and one NNRTI starting
with Atripla in 2006, namely, tenofovir disoproxil fumarate
(TDF), emtricitabine (FTC), and efavirenz (EVF).3 Recent
introductions include Delstrigo, which combines TDF,
lamivudine (3TC), and the NNRTI rilpivirine (RPV), the
first two-drug maintenance therapy Juluca consisting of the
Received: November 20, 2020
Accepted: January 4, 2021
Published: January 6, 2021
INTI dolutegravir (DTG) and RPV, and Dovato (DTG plus
4,5
̈
3TC) for initial therapy in treatment-naive patients.
In addition to discovery of improved drugs in these classes,
there is also much interest in long-acting/extended-release
medications for further gains in patient compliance and
associated prevention of transmission.6 The most widely
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 249−255
249

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
than EFV and RPV. Because the NNRTI allosteric site rather
than an active site is being targeted, inhibition of active sites in
a homologous protein family is not a risk. Indeed, our
compounds along with recent G12C KRAS inhibitors are rare
examples of targeted covalent allosteric inhibitors, which have
enhanced potential for low dosage, low toxicity, and extended
duration of action.16,17 Presently, we report design and
characterization of the first CRTIs that successfully modify
wild-type HIV-1 RT.
The wild-type (WT) protein presents a greater challenge
owing to the lack of cysteine residues in the NNRTI binding
site. There are multiple lysines; however, they are solvent-
exposed and not expected to be adequately nucleophilic.18 The
next choice is the less-explored hydroxyl-containing residues.
In this regard, sulfonyl fluoride and fluorosulfate warheads have
achieved recent successes with serine and tyrosine targets.19−21
In viewing the crystal structure of one of our potent catechol
diether NNRTIs 1 (EC50 = 55 pM) bound to wild-type RT
(Figure 1),7,22 replacement of the chlorine in the central ring
Assays were performed to assess both the in vitro inhibition
of recombinant WT HIV-1 RT and for protection of human
MT-2 T-cells that were infected with the IIIB strain of HIV-1.
The details are unchanged from prior reports.15,22 Briefly, a
PicoGreen-based EnzChek Reverse Transcriptase Assay Kit
(Thermo Fisher Scientific, Inc., E22064) was used to
determine RT activity in the presence of inhibitors in a 96-
well plate format. The enzyme was preincubated with the test
compounds or DMSO control, followed by addition of the
substrate solution including an r(A)350 template and d(T)16
primer. After 30 min of incubation at room temperature,
EDTA was used to quench the reaction. PicoGreen reagent
was then added, and product formation was detected with a
plate reader using excitation and emission at 485 and 520 nm.
The measurements were made in triplicate, and the results
were normalized to DMSO controls to determine IC50 values.
The MT-2 T-cell assay is a standard that has been extensively
applied.7−15 The cells are infected with the IIIB strain of HIV-
1, and the measurements are made in triplicate to yield EC50
values as the dose required to achieve 50% protection of the
infected MT-2 cells and CC50 values for inhibition of MT-2
cell growth by 50%.
The assay results for the 15 compounds in Figure 2 along
with two FDA-approved NNRTI drugs, nevirapine (NEV) and
rilpivirine (RPV), are recorded in Table 1. Some overall
observations are that the trends in the IC50 and EC50 values are
generally similar with a wider range of EC50 results; the IC50
values for the new compounds 3−12 are mostly between those
for NEV and RPV; 6−10 have EC50 values below 100 nM, and
the new compounds, in spite of bearing an electrophilic
warhead, are all less cytotoxic than RPV. Taking a more
detailed look, it may be first noted that 1 is an extraordinarily
potent NNRTI with an IC50 of 3 nM and EC50 of 55 pM.7,22
For the new compounds, we began with 2 and 3 by
considering truncation of the uracilylpropoxy substituent and
placement of the fluorosulfate group either para or meta to the
phenoxy group. However, neither compound showed good
activity, so the subsequent analogues retained the uracil-
containing appendage.
With compound 4, the basic design from Figure 1 was
followed with subtraction of the vinyl group. As the
compounds were being synthesized and assayed, crystal
structures for the complexes with WT RT were being sought
and were eventually obtained for 4−11, as detailed below. The
crystallographic and MS results clearly showed that 4 was the
first CRTI to covalently modify Tyr181 to form the biaryl
sulfate. Under the standard assay procedures, 4 also showed
good potency, with IC50 and EC50 values of 103 and 280 nM.
Additional compounds 6−12 were prepared to evaluate the
effects of modifications to the phenoxy substituent, which
makes aryl−aryl contacts with Tyr188 and Trp229 (Figure 1).
Small changes to the core structure are expected to cause
repositioning that affects the disposition and contact of Tyr181
and the fluorosulfate group. 6−12 turned out to all be potent
inhibitors, with 7, 9, and 10 having sub-100 nM IC50 values,
and the indolizines 8 and 9 having striking EC50 values of 5
and 7 nM. The activity results were surprisingly favorable, as it
was unclear that it would be possible to replace the central
chlorine atom in 1 with the much bulkier and polar
fluorosulfate group without severe loss of potency. In addition
to 4, the crystallographic and MS data show that 11 and 12 are
covalent inhibitors, while the others are noncovalent NNRTIs.
Figure 1. Rendering from the crystal structure of the noncovalent
inhibitor 1 with HIV-1 reverse transcriptase at 2.85 Å resolution
(PDB 4H4M).22 The highlighted distance from the hydroxyl oxygen
atom of Tyr181 to the chlorine atom of 1 is 3.6 Å. All carbon atoms of
ligands are shown in yellow.
with a warhead to attach covalently to Tyr181 arose as a
possibility. Interest focused on the weakly electrophilic
fluorosulfate as the warhead in view of its reported stability
in aqueous solution and lesser off-target reactivity than for
sulfonyl fluorides.20 We ultimately prepared and tested
fluorosulfate-containing analogues of 1 with modifications to
the cyanovinyl and uracil containing appendages (2−13) and
two sulfonyl fluorides (14, 15), as summarized in Figure 2.
The 3-chloro,5-cyanophenoxy substituents, and 1-naphthyloxy
and 8-indolizinyloxy alternatives have all been featured in low-
nM NNRTIs that we have previously reported.7−9
The syntheses of the new compounds are detailed in the
Supporting Information. In each case, the fluorosulfate group
was introduced in good yield by treating a phenol with sulfuryl
fluoride gas and triethylamine in methylene chloride at room
temperature for 2 h.23 The sulfonyl fluorides were prepared
from aryl iodides using a palladium-catalyzed procedure with
1,4-diazabicyclo[2.2.2]octane-bis(sulfur dioxide) (DABSO)
followed by N-fluorobenzenesulfonimide (NFSI) for the
fluorination.24 The identity of assayed compounds was
confirmed by 1H and 13C NMR, high-resolution mass
spectrometry, and ultimately X-ray crystallography for multiple
complexes with WT HIV-1 RT; HPLC analyses established
purity as >95%.
250
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 2. Anti-HIV agents considered here. 1 is a key catechol diether NNRTI; 2−15 are new compounds reported here. 2−13 are potential
CRTIs with a fluorosulfate warhead, and 14−15 have a sulfonyl fluoride warhead. 4, 11, and 12 are shown to be CRTIs.
increased bulk closer to the central ring of the inhibitor leads
to steric conflict with Lys101. 15, the N-methyl analogue of 14,
was also prepared and tested. Although it showed notable
improvement over 14, the IC50 and EC50 results were much
higher than for 4 and 6−10. For 4 and 5, the opposite pattern
was found with the N-methylated 5 being less active, possibly
reflecting a boost from the CRTI character of 4.
Table 1. Measured Activities in the Enzyme Inhibition
(IC50) and Infected T-Cell Assays (EC50), and Cytotoxicity
(CC50)
a
b
c
compd
IC50 (nM)
EC50 (nM)
CC50 (μM)
1
2
3
4
5
6
7
8
9
3.0
4900
833
103
246
82.3
14.7
164
64
0.055
>50000
9200
280
320
58
37
5
7
10
50
78
66
40
62
15
75
55
It may be noted that the three CRTIs (4, 11, 12) all had
similar IC50 values (103−150 nM) and EC50 values (160−280
nM). It is generally expected that covalent inhibitors show
increasing potency with increasing time as the concentration of
free protein declines, while noncovalent inhibition should not
be affected as soon as the binding equilibrium is
achieved.15,16,25 To check this, the dependence of the
enzymatic assay data on incubation time was examined for
1−48 h. For the present compounds, a different pattern was
generally observed with the covalent inhibitors not showing
incubation-time dependence and with the noncovalent ones
showing loss of potency over time. For example, the measured
IC50 for 4 was 103 nM with the usual 1 h incubation and it was
163 nM after a 48 h incubation, while the corresponding
results for 8 were 164 and 1115 nM, and for 9 they were 64
and 429 nM. Though further kinetic analyses are warranted, a
possible explanation is that there is some degradation of the
inhibitors over the 2 days as reflected in the increasing IC50
values for the noncovalent inhibitors, while this effect is being
offset by the covalent inhibitors removing free protein. A few
compounds were also tested for inhibition of recombinant RT
protein bearing the clinically common Tyr181Cys mutation.
For the indolizines 8, 9, and 10, very similar IC50 values were
obtained for the WT (164, 64, 42 nM) and mutant (202, 63,
41 nM) proteins, respectively, while the CRTI 4 showed
greater inhibitory potency for the variant (45 nM) than the
10
11
12
13
14
15
nevirapine
rilpivirine
42.4
150
119
ND
5000
375
1060
38
26
26
42
45
100
90
60
>100
8
270
160
14000
17000
2700
110
0.67
a
b
For 50% inhibition of reverse transcription (nM). For 50%
c
protection in MT-2 cells (nM). For 50% inhibition of MT-2 cell
growth (μM).
A few more compounds were prepared. Model building of
structures indicated that both meta and para isomers as for 2
and 3 might be viable. Thus, the meta analogue 13 of the CRTI
4 was synthesized, but it was found to be a comparatively weak
NNRTI with an EC50 of 14 μM. The sulfonyl fluoride 14
corresponding to the fluorosulfate 4 was also prepared and
found to be a weak inhibitor; modeling suggests that the
251
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 3. Renderings from the 2.70 Å crystal structure for 4 covalently bound to wild-type HIV-1 reverse transcriptase. (A) Closeup highlighting
the covalent bond between the oxygen atom of Tyr181 and the sulfate sulfur atom of 4. (B) View highlighting three hydrogen bonds between the
backbone and side chain nitrogen atoms of Lys101 and two sulfate oxygen atoms (PDB 7KRD).
WT enzyme (82 nM). These results are encouraging, and
further study of resistance profiles is of interest, but beyond the
present focus.
Phe227 triangle are also expanded slightly to 8.7, 4.9, and 8.7
Å.
As a further example of a covalent complex, the structure for
12 is provided in Figure 5A. The side chains for Lys101,
For the crystallography, recombinant WT RT52A enzyme
was used with cocrystallization methods, as previously
described.8−10,15,22 Structures were obtained for the complexes
with 4−12 at resolutions of 2.60−2.73 Å. The first covalent
structure was obtained for 4, as illustrated in Figure 3. The
covalent bond between the Tyr181 oxygen atom and the sulfur
of the warhead is 1.5 Å, and there are hydrogen bonds for both
the side chain and backbone nitrogen atoms of Lys101 with
sulfate oxygen atoms. There is also a hydrogen bond between
an uracil oxygen atom and the backbone nitrogen atom of
Lys103. The distortion of the binding site is slight in
comparison with related noncovalent structures. For example,
the distances between the Cβ atoms for the triangle made by
the key aromatic residues Tyr188−Trp229−Phe227 are 8.4,
4.7, and 8.5 Å for the structure with 4 (Figure 3) and 8.5, 5.7,
and 9.3 for 1 (Figure 1).
As an example of a structure for one of the noncovalent
inhibitors with an intact fluorosulfate group, the complex for 6
bound to WT RT is illustrated in Figure 4. In this case, the
distance between the tyrosine oxygen and sulfur atoms is 3.8 Å
and the shortest contact between the backbone nitrogen atom
of Lys101 and an oxygen atom of the warhead is 4.0 Å versus
3.4 Å for 4. The Cβ−Cβ distances for the Tyr188−Trp229−
Figure 5. (A) Rendering from the 2.63 Å crystal structure for 12
covalently bound to wild-type HIV-1 reverse transcriptase. Note the
network of hydrogen bonds for the sulfate group with the ammonium
groups of Lys101 and Lys103 and the backbone NH of Lys101 (PDB
7KRC). (B) The three conformations of dimethylsulfate.
Lys102, and Lys103 are particularly well resolved in this
structure and reveal a striking hydrogen-bonded network for
the ammonium groups of Lys101 and Lys103 and the
backbone nitrogen of Lys 101 with all four oxygens of the
sulfate group. In both this structure and the one for 11 (not
illustrated), the covalent bond length is 1.7 Å between the
tyrosine oxygen atom and the sulfur atom of the sulfate group.
Again, there is no significant distortion of the binding site as
reflected in the Cβ−Cβ distances for the Tyr188−Trp229−
Phe227 triangle of 8.4, 4.7, and 8.5 Å. Furthermore, concerning
the conformation of the diphenyl sulfate substructure, three
conformational energy minima are normally expected for
dialkyl or diaryl sulfates corresponding to G(gauche)G,
GT(trans), and TT for the two C−O−S−O dihedral angles
with relative energies of ca. 0.0, 1.0, and 3.0 kcal/mol,
respectively (Figure 5B).26 With use of the BOSS program and
OPLS/CM1A force field, these results are well reproduced.27,28
For the covalent complexes of 4, 11, and 12, the inhibitor C−
O−S−O dihedral angle is G; however, the Tyr181 C−O−S−
O dihedral angle is closer to syn at ca. 10°. Thus, there is some
strain relative to a GG conformation; it is roughly estimated to
Figure 4. Rendering from the 2.73 Å crystal structure for 6
noncovalently bound to wild-type HIV-1 reverse transcriptase. The
highlighted distance between the Tyr181 oxygen atom and
fluorosulfate sulfur atom is 3.8 Å (PDB 7KRE).
252
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 6. Representative spectra for ESI TOF MS analysis of RT covalent catalysis. (A) (top) spectra for RT p66 and p51 subunits; (bottom)
spectra of RT p66 and p51 subunits after incubation with 11. (B) Summary of calculated and observed masses for RT with 4, 11, and 12.
be 5 kcal/mol with the OPLS/CM1A force field from
constrained energy-minimizations for diphenyl sulfate. Such
strain is readily offset by formation of the new covalent O−S
covalent bond and elimination of the fluoride ion. Far more
strained aryl sulfates and sulfonates have been well studied
such as catechol sulfate.29
inhibition of the wild-type protein is reported by taking
advantage of the NNRTI binding site and modification of
known noncovalent inhibitors with introduction of an
electrophilic warhead. As there are no cysteine residues in
the vicinity of the binding site, the less precedented covalent
modification of a tyrosine, specifically Tyr181, was pursued.
Success was demonstrated through protein crystallography and
mass spectrometry for three compounds, 4, 11, and 12, which
incorporate a fluorosulfate warhead. These compounds and six
analogues were found to be nanomolar inhibitors of HIV-RT
through both an in vitro enzyme-inhibition assay and an
infected T-cell assay. Further work is investigating potential
ramifications of the covalent inhibition for induction of
resistance and considering additional residues as targets in
the NNRTI binding site.
The crystallographic conclusions on the covalent modifica-
tion of Tyr181 by 4, 11, and 12 were fully supported by
electrospray ionization−time-of-flight mass spectrometry (ESI-
TOF MS), which was used to determine the molecular weight
of the intact protein and to examine potential covalent
modification reflected by increases in mass. The same
procedures were followed as in the previous work.15 WT RT
20 μM was incubated alone or with 0.5 mM inhibitor in 1.5%
DMSO for 6 days at 4 °C. Samples were prepared by reverse-
phase HPLC and injected into a Bruker IMPACT II ESI-TOF
mass spectrometer (Bruker Inc., Billerica, MA) operated in
positive ion mode. As shown in Figure 6A, the top two panels
show a reference sample of WT RT with peaks at 63 907.5 and
50 027.8 Da, corresponding to the p51 and p66 subunits of
RT. The bottom panel shows representative spectra after the
incubation of RT with 11. As p66 contains the catalytic and
NNRTI binding sites, a mass shift for only this subunit would
be expected upon introduction of the covalent inhibitors. As
illustrated in this bottom panel, no shift is observed for the p51
subunit. For spectra showing the p66 subunit, a peak
corresponding to the unmodified p66 is observed at 63 097.5
as well an additional peak at 64 378.8 with a mass shift of 471
corresponding to the covalent addition of 11. Similarly, peaks
are observed at 64 370 (mass shift of 461) for 4 and 64 395.5
(mass shift 487) for 12. The calculated and observed masses
for each p66 and p51 species are summarized in Figure 6B.
In summary, a new class of anti-HIV agents, which
covalently modify HIV-1 reverse transcriptase is under
investigation. In this work, the first successful covalent
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00612.
Details on the syntheses and characterization of all new
compounds and on the crystallography for complexes of
HIV-1 RT with 4, 6, 11, and 12 (PDF)
X-ray data collection results (XLSX)
Accession Codes
The crystal structures have been deposited in the RCSB
Protein Data Bank with the PDB codes 7KRD, 7KRE, 7KRF,
and 7KRC, respectively.
AUTHOR INFORMATION
■
Corresponding Authors
William L. Jorgensen − Department of Chemistry, Yale
University, New Haven, Connecticut 06520-8107, United
253
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
States; orcid.org/0000-0002-3993-9520;
Email: william.jorgensen@yale.edu
National Laboratory under Contract No. DESC0012704. The
Life Science Biomedical Technology Research resource is
primarily supported by the NIH, NIGMS through a
Biomedical Technology Research Resource P41 grant (P41
GM111244), and by the DOE Office of Biological and
Environmental Research (KP1605010).
Karen S. Anderson − Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut
06520-8066, United States; Department of Molecular
Biophysics and Biochemistry, Yale University School of
Medicine, New Haven, Connecticut 06520-8066, United
States; orcid.org/0000-0003-3433-0780;
REFERENCES
■
Email: karen.anderson@yale.edu
(1) Frank, T. D.; et al. Estimates of global, regional, and national
incidence, prevalence, and mortality of HIV, 1980−2017, and
Forecasts to 2030. Lancet HIV 2019, 6, e831−e859.
(2) De Clercq, E.; Li, G. Approved Antiviral Drugs over the Past 50
Years. Clin. Microbiol. Rev. 2016, 29, 695−747.
Authors
Joseph A. Ippolito − Department of Chemistry, Yale
University, New Haven, Connecticut 06520-8107, United
States; Department of Pharmacology, Yale University School
of Medicine, New Haven, Connecticut 06520-8066, United
States
(3) Sebaaly, J. C.; Kelley, D. Single-Tablet Regimens for the
Treatment of HIV-1 Infection. Ann. Pharmacother. 2017, 51, 332−
344.
Haichan Niu − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
Nicole Bertoletti − Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut
06520-8066, United States; orcid.org/0000-0001-5167-
402X
Zachary J. Carter − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
Shengyan Jin − Department of Pharmacology, Yale University
School of Medicine, New Haven, Connecticut 06520-8066,
United States
(4) Wang, Y.; De Clercq, E.; Li, G. The Current and Emerging
Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) for HIV-
1 Treatment. Expert Opin. Drug Metab. Toxicol. 2019, 15, 813−829.
(5) Sancar, F. Two-Drug Regimen for HIV. JAMA 2019, 321, 1862.
(6) Gulick, R. M.; Flexner, C. Long-Acting HIV Drugs for
Treatment and Preventation. Annu. Rev. Med. 2019, 70, 137−150.
(7) Bollini, M.; Domaoal, R. A.; Thakur, V. V.; Gallardo-Macias, R.;
Spasov, K. A.; Anderson, K. S.; Jorgensen, W. L. Computationally-
Guided Optimization of a Docking Hit to Yield Catechol Diethers as
Potent Ani-HIV Agents. J. Med. Chem. 2011, 54, 8582−8591.
(8) Lee, W.-G.; Gallardo-Macias, R.; Frey, K. M.; Spasov, K. A.;
Bollini, M.; Anderson, K. S.; Jorgensen, W. L. Picomolar Inhibitors of
HIV Reverse Transcriptase Featuring Bicyclic Replacement of a
Cyanovinylphenyl Group. J. Am. Chem. Soc. 2013, 135, 16705−16713.
(9) Lee, W.-G.; Gallardo-Macias, R.; Frey, K. M.; Spasov, K. A.;
Bollini, M.; Anderson, K. S.; Jorgensen, W. L. Picomolar Inhibitors of
HIV Reverse Transcriptase: Design and Crystallography of Naphthyl
Phenyl Ethers. ACS Med. Chem. Lett. 2014, 5, 1259−1262.
(10) Lee, W. G.; Chan, A. H.; Spasov, K. A.; Anderson, K. A.;
Jorgensen, W. L. Design, Conformation, and Crystallography of 2-
Naphthyl Phenyl Ethers as Potent Anti-HIV Agents. ACS Med. Chem.
Lett. 2016, 7, 1156−1160.
Krasimir A. Spasov − Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut
06520-8066, United States
́
Jose A. Cisneros − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
Margarita Valhondo − Department of Chemistry, Yale
University, New Haven, Connecticut 06520-8107, United
States
Kara J. Cutrona − Department of Chemistry, Yale University,
New Haven, Connecticut 06520-8107, United States
(11) Jorgensen, W. L. Computer-aided Discovery of Anti-HIV
Agents. Bioorg. Med. Chem. 2016, 24, 4768−4778.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00612
(12) Kudalkar, S. N.; Beloor, J.; Chan, A. H.; Lee, W. G.; Jorgensen,
W. L.; Kumar, P.; Anderson, K. S. Structural and Preclinical Studies of
Computationally Designed Non-Nucleoside Reverse Transcriptase
Inhibitors for Treating HIV infection. Mol. Pharmacol. 2017, 91, 383−
391.
(13) Kudalkar, S. N.; Beloor, J.; Quijano, E.; Spasov, K. A.; Lee, W.-
G.; Cisneros, J. A.; Saltzman, W. M.; Kumar, P.; Jorgensen, W. L.;
Anderson, K. S. From In Silico Hit to Long-Acting Late-Stage
Preclinical Candidate to Combat HIV Infection. Proc. Natl. Acad. Sci.
U. S. A. 2018, 115, E802−E811.
(14) Kudalkar, S. N.; Ullah, I.; Bertoletti, N.; Mandl, H. K.;
Cisneros, J. A.; Beloor, J.; Chan, A. H.; Quijano, E.; Saltzman, W. M.;
Jorgensen, W. L.; Kumar, P.; Anderson, K. S. Structural and
Pharmacological Evaluation of a Novel Non-nucleoside Reverse
Transcriptase Inhibitor as a Promising Long Acting Nanoformulation
for Treating HIV. Antiviral Res. 2019, 167, 110−116.
(15) Chan, A. H.; Lee, W.-G.; Spasov, K. A.; Cisneros, J. A.;
Kudalkar, S. N.; Petrova, Z. O.; Buckingham, A. B.; Anderson, K. S.;
Jorgensen, W. L. Covalent inhibitors for eradication of drug-resistant
HIV-1 reverse transcriptase: from design to protein crystallography.
Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 9725−9730.
Author Contributions
∥J.A.I., H.N., and N.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Gratitude is expressed to the National Institutes of Health
(AI44616, GM32136, GM49551, AI155072) for research
support. Crystal screening was conducted with support in
the Yale Macromolecular X-ray Core Facility
(1S10OD018007-01). This work is based upon research
conducted at the Northeastern Collaborative Access Team
beamlines, which are funded by the National Institute of
General Medical Sciences from the National Institutes of
Health (P41 GM103403). This research used resources of the
Advanced Photon Source, a U.S. Department of Energy
(DOE) Office of Science User Facility operated for the DOE
Office of Science by Argonne National Laboratory under
Contract No. DE-AC02−06CH11357. This research used the
FMX beamline of the National Synchrotron Light Source II, a
U.S. Department of Energy (DOE) Office of Science User
Facility operated for the DOE Office of Science by Brookhaven
(16) Nussinov, R.; Tsai, C.-J. The Design of Covalent Allosteric
Drugs. Annu. Rev. Pharmacol. Toxicol. 2015, 55, 249−267.
(17) McCormick, F. Sticking It to KRAS: Covalent Inhibitors Enter
the Clinic. Cancer Cell 2020, 37, 3−4.
(18) Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting
Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56, 15200−15209.
254
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(19) Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.;
Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.;
Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W.
Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s)
through Chemoselective SuFFex Reaction with a Binding Site Tyr
Residue. J. Am. Chem. Soc. 2016, 138, 7353−7364.
(20) Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.;
Hett, E. C.; Kyne, R.E., Jr.; Robinson, R. P.; Jones, L. H. Covalent
Enzyme Inhibition through Fluorosulfate Modification of a Non-
catalytic Serine Residue. ACS Chem. Biol. 2017, 12, 2015−2020.
(21) Jones, L. H. Emerging Utility of Fluorosulfate Chemical Probes.
ACS Med. Chem. Lett. 2018, 9, 584−586.
(22) Frey, K. M.; Bollini, M.; Mislak, A. C.; Cisneros, J. A.; Gallardo-
Macias, R.; Jorgensen, W. L.; Anderson, K. S. Crystal Structures of
HIV-1 Reverse Transcriptase with Picomolar Inhibitors Reveal Key
Interactions for Drug Design. J. Am. Chem. Soc. 2012, 134, 19501−
19503.
(23) Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI)
Fluoride Exchange (SuFEx): Another Good Reaction for Click
Chemsitry. Angew. Chem., Int. Ed. 2014, 53, 9430−9448.
(24) Davies, A. T.; Curto, J. M.; Bagley, S. W.; Willis, M. C. One-pot
Palladium-catalyzed Synthesis of Sulfonyl Fluorides for Aryl
Bromides. Chem. Sci. 2017, 8, 1233−1237.
(25) De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.;
Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med.
Chem. 2017, 138, 96−114.
́
(26) Borba, A.; Gomez-Zavaglia, A.; Simoes, P. N. N. L.; Fausto, R.
̃
Matrix-isolation FT-IR Spectra and Theoretical study of Dimethyl
Sulfate. Spectrochim. Acta, Part A 2005, 61, 1461−1470.
(27) Jorgensen, W. L.; Tirado-Rives, J. Potential Energy Functions
for Atomic-Level Simulations of Water and Organic and Biomolecular
Systems. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 6665−6670.
(28) Dodda, L. S.; Cabeza de Vaca, I.; Tirado-Rives, J.; Jorgensen,
W. L. LigParGen Web Server: An Automatic OPLS-AA Parameter
Generator for Organic Ligands. Nucleic Acids Res. 2017, 45, W331−
W336.
(29) Kaiser, E. T. Enzymatic and Nonenzymatic Reactions of Cyclic
Sulfonate and Sulfate Esters. Acc. Chem. Res. 1970, 3, 145−151.
255
https://dx.doi.org/10.1021/acsmedchemlett.0c00612
ACS Med. Chem. Lett. 2021, 12, 249−255